Osteoarthritis (OA) is a degenerative cartilage disease that affects joints, causing pain and disability. Conservative treatments, such as physiotherapy or non-steroidal anti-inflammatory drugs, are possible during the early phase of OA, but their efficacy is limited. Besides, OA’s last stage is characterized by unbearable pain and massive cartilage disruption, thus requiring a surgery solution .The gap between these two phases is huge, but there are no definitive solutions for a complete symptomatic resolution. Intra-articular administration of hyaluronic acid, as well as platelet-rich-plasma (PRP) treatments, which exploit the use of autologous growth factors , can offRegenerative medicine can be a conservative and optimal solution for OA treatment. Mesenchymal stem cells (MSCs) can release growth factors and anti-inflammatory cytokines that promote tissue regeneration . MSCs caAdipose-derived MSCs (AD-MSCs) can be obtained by using medical devices that comply with minimal manipulation requirements set by European and United States regulatory agencies, and many authors have demonstrated their safety and efficacy –15. Howe2, and willingness to be followed up for nine months. Patients with a severe knee varus/valgus deformity , an autoimmune disease, or a rheumatic illness were excluded. All patients signed an informed consent form to participate in the study, which followed the ethical standards of the 1964 Helsinki Declaration and its later amendments.This was an analysis of patients with symptomatic knee OA who underwent an intra-articular injection of purified adipose tissue between March 2018 and July 2019 at two centers in Madrid: Maestranza Medical Center and Beata María Ana Hospital. Individuals aged between 40 and 80 years old, diagnosed with unilateral or bilateral knee OA according to the American College of Rheumatology criteria  and the All patients underwent minor liposuction to collect abdominal adipose tissue. The procedure consisted of infiltration of Klein solution  into the abdominal subcutaneous fat, followed by liposuction with fenestrated blunt cannulae and collection of approximately 60 mL of lipoaspirated fat. Then, the lipoaspirate was processed with Lipocell , a Class II-a medical device, following the manufacturer’s instructions. Briefly, the lipoaspirate was introduced in the device, where it was dialyzed with a filter and washed with 300 mL-500 mL of NaCl 0.9%. In roughly 5 min, this device allows to eliminate undesired products while preserving AD-MSCs viability and doubling the AD-MSCs yield with low manipulation . SubsequPatients were discharged the same day of the infiltration. If necessary, the pain was controlled with ice and acetaminophen; no ibuprofen or any other NSAIDs were allowed. Elevation and flexion of the knee were recommended since the first day, and partial weight-bearing with the help of crutches was suggested for two weeks, always depending on the patient’s pain. Afterward, patients began to perform eccentric exercises supervised by a physiotherapist for one month. Low-impact sports  and high-impact physical activity were allowed six-to-eight weeks and three months after the procedure, respectively.The analyzed variables included age, sex, BMI, and Kellgren-Lawrence classification of OA. In addition, the VAS score and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) were used to assess pain and functionality, respectively, before the procedure and at three, six, and nine months after treatment.Categorical variables were presented as frequency and percentages, and quantitative variables as the mean and standard deviation (SD).WOMAC and VAS score differences among the different times evaluated were assessed using the Wilcoxon signed-rank test. The threshold for statistical significance was set at a two-sided alpha value of 0.05. All analyses were performed with GraphPad Prism v.9.2. According to the Kellgren-Lawrence classification, 3 (12.5%), 10 (41.7%), and 11 (45.8%) patients presented Grade 2, Grade 3, and Grade 4 OA, respectively. In addition, 14 (58.3%) patients underwent knee arthroscopy for lavage and debridement of loose bodies before injecting the purified lipoaspirated fat.A total of 24 patients were enrolled, 15 (62.5%) women and 9 (37.5%) men, with a mean (SD) of 63.8 (11.4) years and a mean (SD) BMI of 28.3 (5.1) kg/mPatients’ pain decreased progressively over time, with a VAS mean (SD) score of 8.3 1.0) before treatment, and 6.5 (1.4), 5.1 (1.9), and 4.5 2.1) at three, six, and nine months after treatment, respectively  that promote tissue regeneration , 20; on In this analysis, the treatment is effective up until nine months. This improvement could last even longer, as reported in other studies , 14, butThis work has some limitations. First, it lacks a control group and the radiological assessment after the procedure. Indeed, future studies should compare the results of adipose tissue infiltration with those of traditional treatments  and include radiological evaluations post-treatment. In addition, the analysis did not allow to differentiate the outcomes of adipose tissue infiltration according to the performance of a previous knee arthroscopy or the degree of knee OA. Although these subanalyses may have added more value to the results, they were not deemed appropriate due to the small sample size. Further studies with more patients would be of interest in order to assess the impact of the degree of OA and concomitant treatments on the pain and functionality improvement of adipose tissue infiltration. However, the aim of this study was not to assess separately the efficacy of Lipocell according to OA degrees, but to avoid the exclusion of patients based on this variable, as happens in most studies. In addition, a large proportion of patients presented with grade IV OA. Therefore, the results add evidence to the existing literature on autologous adipose tissue infiltration with Lipocell device as well as providing data about patients not usually assessed.Joint rejuvenation is key to fight OA, and regenerative medicine can help to fill the gap between conservative treatments and joint replacement. Moreover, patients are not often willing to undergo surgery, but they are good with less invasive procedures, such as injections. In the future, if regulatory approval is obtained, allogenic and expanded MSCs could be used for the preparation of precise cell doses in vials without requiring the harvest on every single patient. However, at the moment, regulations impose some restrictions, and people could be reluctant to receive donors’ cells.In conclusion, this study supports the safety and efficacy of the intra-articular infiltration of purified autologous adipose tissue in OA patients. If more studies confirm these findings, maybe in a few years it could become state of the art in early OA treatment.


In this study we have proved that dogs, pigs, cattle, and horses, species belonging to four distinct families, differ in regard to microscopical characteristics of their C thyrocytes. Although the total number of C thyrocyte profiles and their localization within thyroid lobes were comparable, each of the examined species displayed their unique morphological characteristics and distribution pattern. The differences described in our study, easily recognizable microscopically, can be used as a reference material for further studies focused on C thyrocytes biology in physiological and pathological conditions.The number, morphology, and distribution of C thyrocytes within the thyroid gland vary among species; however, studies in domestic animals are limited. In this study we compared the morphology, distribution pattern, and percentage of C thyrocytes in four domestic species: dogs, pigs, horses, and cattle. Eighty thyroid glands, 20 per species, were examined. C thyrocytes were visualized immunohistochemically with anti-calcitonin rabbit polyclonal antibody alone and combined with the periodic acid Schiff method to simultaneously visualize C thyrocytes with the basement membranes of thyroid follicles. C thyrocyte morphology varied considerably between species, from oval- (dogs) and spindle-shaped (pigs) to polymorphic (cattle and horses). Bovine C thyrocytes demonstrated cytoplasmic protrusion. C thyrocytes were located intrafolliculary , epifollicularly , or interfolicularly (cattle). Most porcine and bovine C thyrocytes existed individually whereas canine C thyrocytes usually formed clusters. In horses, they tended to form groups of various shapes and sizes or even rims encompassing whole follicles. In all species, the number of C thyrocyte profiles increased from the periphery to the central area of the thyroid lobe. The mean total fraction of C thyrocytes in the superficial, intermediate, and central areas were as follows: 2.55%, 8.43%, and 12.48% in dogs; 3.81%, 7.66%, and 10.79% in pigs; 1.55%, 7.44%, and 8.87% in horses; and 2.62%, 10.75%, and 12.96% in cattle. No statistical differences in the total number of C thyrocyte profiles were observed among species . Our results indicated that the studied species displayed their own morphological characteristics and distribution pattern of C thyrocytes; however, total numbers of C thyrocyte profiles and their localization within the thyroid lobe are comparable. The thyroid gland contains two types of endocrine cells: T thyrocytes  and C thyrocytes ; however, C thyrocytes constitute only a minor proportion of the total thyroid parenchyma. The cells are responsible for maintaining calcium homeostasis by managing calcitonin production, as well as the release of various regulatory peptides involved in the local regulation of T thyrocyte activity .Although the number, morphology, and distribution of C thyrocytes within the thyroid gland are known to vary between species ,2, detaiMost studies conducted on canine C thyrocytes have focused on C thyrocytes complexes ,16,17; tSome information on equine C thyrocyte morphology is given by Blähser , TanimurBos taurus), dogs (Canis familiaris), horses , and pigs (Sus scrofa domesticus).No studies so far appear to have examined the morphology and localization of C thyrocytes in cattle. Moreover, no studies describe the localization of C thyrocytes in relation to thyroid follicles. To date, the positioning of C thyrocytes was only estimated based on their localization relative to T thyrocytes without the visualization of the basement membranes of thyroid follicles. The comparative histological studies subjected on C thyrocytes focused mainly on rodents ,6 and upEighty thyroid glands were included in the study, twenty per species. The following groups of animals were examined: dogs , pigs , horses , and cattle .The study complied with Directive 2010/63/EU and the Act of the Polish Parliament dated 15 January 2015 on the protection of animals used for scientific purposes . None of the animals were killed for the study: all tissue specimens were collected from animal carcasses. The thyroid glands originating from cattle, horses, and pigs were collected from freshly slaughtered animals. The canine thyroid glands were obtained during necropsy from animals euthanized in veterinary clinics due to illnesses unrelated to thyroid function, such as paraparesis, aggression, car accidents, cardiorespiratory or renal failure, or tumors of the skin or abdominal organs.Only thyroid glands without any macroscopic lesions were included. In each case, the central part of the left thyroid lobe, sectioned transversely, was collected. We chose this part of the thyroid lobe, as according to available data in this region the C thyrocytes are the most numerous ,29,30. TThe C thyrocytes were visualized immunohistochemically using an anti-calcitonin rabbit polyclonal antibody ; cross-reactivity for each examined species has been confirmed by the manufacturer and in previous studies ,25,26,27Moreover, for each species, the C thyrocytes were co-visualized with the basement membranes of the thyroid follicles. Briefly, five additional slides per species representing the thyroid gland sections with the best visualized C thyrocytes were selected. After immunohistochemical detection with anti-calcitonin antibody, these specimens were subjected to further staining by the periodic acid Schiff method (own modification related to the use of these two methods in one staining procedure). Briefly, the reaction with the primary antibody was visualized using the DAB solution (Agilent Dako), following which, the slides were rinsed with tap water for 10 min and placed in distilled water for 5 min. They were then subjected to staining by the routine periodic acid Schiff method . The sliThe C thyrocytes were quantified in three separate areas of the medial region of the thyroid lobe: superficial, central, and intermediate. The cells were counted manually in 14 randomly selected visual fields per each area (154.89 × 193.61 µm) at a microscope magnification of 600×. The visual fields were located uniformly throughout each area of interest. In each visual field, the overall number of C and T thyrocyte profiles were calculated. Following this, the results from each area were summarized, and the proportion of C thyrocyte profiles to profiles of total endocrine cell population (fraction of C thyrocytes) was calculated according to the following formula:In each case, the fraction of C thyrocytes was scored in both tissue sections originating from each lobe and the mean value was calculated. The numbers were recorded for each area of the thyroid lobe and for the whole tissue section .p ≤ 0.05 was considered significant.The obtained data were analyzed using Statistica 13.3 for Windows . All were presented as mean values ± SD, differences in C thyrocyte distribution patterns were calculated using the Mann–Whitney U-test. Fractions of C thyrocytes were compared between the three areas of the thyroid lobe and between species. C thyrocyte morphology varied considerably between species. In all examined species, the cellular outlines of C thyrocytes were clearly visible in individual cells but usually faded within cell clusters, especially in horses.In dogs, the C thyrocytes were oval or polygonal in shape, with centrally-located oval or slightly irregular nuclei c–e.In pigs, the C thyrocytes had a spindle shape; these were strongly elongated, with an oval nucleus located eccentrically in most cases g,h.The equine C thyrocytes were polymorphic, ranging in shape from oval or polygonal to elongated or irregular. The nuclei were round or oval, depending on cell shape, and located centrally h–j. In sIn cattle, the C thyrocytes were also polymorphic, being polygonal, oval, or irregular in shape. Some possessed clearly-visible long cytoplasmic processes g–i; thesIn all examined species, the C thyrocytes were not uniformly distributed throughout the thyroid gland parenchyma. In the narrow subcapsular region of the thyroid gland, only single C thyrocytes were observed. In deeper regions, the C thyrocytes were located unevenly: areas of thyroid gland parenchyma containing C thyrocytes were surrounded by areas either completely lacking C thyrocytes or containing only individual C thyrocytes a,b. The The location of C thyrocytes in relation to the thyroid follicles differed between species. In the cattle specimens, the C thyrocytes were most likely in contact with the colloid, as some cells were observed to reach the apical surface of the T thyrocytes c. No sucIn dogs, the C thyrocytes mostly formed groups of 2–10 cells; however, some larger groups of approximately 10–20 cells were observed, with some exceeding 20 cells. Individual C thyrocytes were rare . The C tIn pigs, the C thyrocytes were present exclusively intrafollicularly and were observed to adhere to the follicular basement membrane . Most ofIn horses, the C thyrocytes tended to form “chains” d,e or grIn cattle, the vast majority of C thyrocytes were located individually a,b; howeFor almost all cases taken from the four species included in the study, the highest fractions of C thyrocytes were observed in the central area of the medial part of the thyroid lobe, as viewed in the cross-section, followed by the intermediate and superficial areas. p ≤ 0.001). However, taking into account individual cases, the percentage differences in C thyrocyte profiles levels between the intermediate and central areas were less than 1% in three of 20 cases, and less than 2% in another two of 20 cases. In two of 20 cases, no significant differences were found between the superficial, central, and intermediate areas. The fraction of C thyrocytes in three different regions of the medial part of the canine thyroid lobe and the total fraction of C thyrocytes in particular cases are shown in In the dog specimens, the mean fraction of C thyrocytes was 2.55 ± 5.32 in the superficial area, followed by 8.43 ± 9.29 in the intermediate area, and 12.48 ± 9.42 in the central area . All thep ≤ 0.001)  . The difp ≤ 0.001)  . In thisp ≤ 0.001). In three of the 20 cases, the fraction of C thyrocytes was higher in the intermediate area than the central area; this difference was less than 1% in the two cases, and greater than 2% in the other case. Among the remaining 17 of 20 cases, the difference in C thyrocyte profiles percentage between the central and intermediate areas was less than 2% in six animals. The fraction of C thyrocytes in three different regions of the medial part of the bovine thyroid lobe and the total fraction of C thyrocytes in particular cases are shown in In the cattle specimens, the mean fraction of C thyrocytes was 2.62 ± 4.70, 10.75 ± 7.43, and 12.96 ± 7.97 for the superficial, intermediate, and central areas, respectively . These dComparable mean total fractions of C thyrocytes were observed for the whole examined tissue section, in dogs (mean 8.87 ± 3.72), cattle (mean 8.58 ± 2.08), and pigs (mean 7.98 ± 2.94). A slightly different value was obtained in horses, i.e., 5.83 ± 2.56, however, this difference was not significant .This study gives the first detailed comparison of the morphological characteristics of C thyrocytes in four species belonging to the Canidae, Suidae, Bovidae, and Equidae. All species were found to demonstrate unique C thyrocyte morphology and distribution patterns that can be easily recognized microscopically.Our observations of canine C thyrocytes confirm those from previous studies, particularly that C thyrocytes are usually clustered together in this species ,7,9. HowSome discrepancies exist regarding the morphology of C thyrocytes in pigs. In most cases, they are described as elongated ,6,11, whOur results clarified the discrepancy regarding the distribution of porcine C thyrocytes. Some authors  observedOnly limited data exist regarding the morphology of equine C thyrocytes. The most informative paper, in our opinion, was a methodological study by Blähser  focusingBlähser  and TaniSome discrepancies exist regarding the localization of C thyrocytes in horses. While Blähser  indicateEven less is known about C thyrocytes in cattle. A review by Kameda  reports A characteristic morphological feature of the bovine C thyrocytes observed in the present study was the presence of obvious, long cytoplasmic processes, which have not been reported in previous studies of this species ,35. HoweIn our present study, the bovine C thyrocytes were found to be located mainly individually or, less frequently, in small groups consisting of two to five cells. A similar distribution has been noted in sheep ; howeverThe specimens were also subjected to combined staining to visualize simultaneously the C thyrocytes and the basement membrane of thyroid follicles. This is the first such co-visualization performed in domestic animals. Previous studies have estimated the localization of C thyrocytes only based on their positioning in relation to T thyrocytes and stromal elements. The localization of the cells was reported according to Sawicki and Kuczyński . Only inSome discrepancies exist between studies regarding the location of C thyrocytes in relation to thyroid follicles in dogs. While some studies indicate that C thyrocytes are located exclusively outside the follicular wall, in the parafollicular and interfollicular positions ,7, Rost Fetter and Capen  report tDiscrepancies exist regarding the localization of C thyrocytes in horses. They have been found to be present within the thyroid follicle wall in some studies  and to bLittle is known about the location of bovine C thyrocytes in relation to thyroid follicles; however, our present findings indicate they can occur in three positions: intrafollicular, epifollicular, and interfollicular. While previous study has also confirmed intrafollicular arrangement , the preIn our study, C thyrocytes were observed in three positions: intra-, epi-, and interfollicularly, with the first one being the most common. The intrafollicular localization of C thyrocytes occurred in all analyzed species. The second most frequent localization of C thyrocytes was the epifollicular position presented in all species except pigs, whereas interfollicularly located C thyrocytes were found only in cattle. In contrast, the interfollicular localization of C thyrocytes is frequent in humans. C thyrocytes in this localization are apparently more common than intrafollicular; the second localization of C thyrocytes described in humans ,39,40,41The location of C thyrocytes within the thyroid lobe parenchyma is not uniform, being located primarily in the center of the thyroid lobe, together with the highest density of active follicles. This is doubtlessly associated with the fact that C thyrocytes are involved in the intrathyroidal regulation of T thyrocyte function by the secretion of various regulatory factors . In contTo date, differences in C thyrocytes distribution within the thyroid parenchyma have been reported in rodent species, pigs, horses, and European bison ,10,13,15Regarding the percentage of C thyrocyte profiles in relation to the profiles of total endocrine cell population, dogs, pigs, and cattle demonstrated similar total fractions of C thyrocytes (approximately 8–9%), and these were only slightly higher than in horses (approximately 6%). These results are in line with previous reports in other species, i.e., ranging from less than 1% in humans ,5,42. OnUntil now, dogs were considered to have the highest percentage of C thyrocytes among all animal species . HoweverIn pigs, the percentage of C thyrocytes has been found to vary from approximately 1%  to 4% 1. Our resOur findings are the first to provide fractions of C thyrocytes in horses and cattle. Only one previous study has analyzed the percentage of C thyrocytes in European bison . While EOur results, although based on the number of C thyrocyte profiles calculated only in the medial part of the thyroid lobe, indicate that the differences in the percentage of C thyrocytes within the thyroid gland are not pronounced and that most extensive inter-species discrepancies are probably associated with the method of calculation used in studies and the choice of thyroid gland area. Both our present work and that on European bison  were perSome studies indicate that the shapes, sizes, and distribution patterns of C thyrocytes are species-specific ,15,22. IThe study serves detailed microscopic characteristics of C thyrocytes in healthy animals. It can be valuable for further studies of C thyrocytes, especially in terms of their paracrine functions. Moreover, the description of normal C thyrocytes morphology and distribution pattern can also serve as a reference for further microscopic studies on C thyrocytes in pathological conditions, such as C thyrocyte hyperplasia or medullary carcinoma.Canidae, Suidae, Bovidae, and Equidae, carried their unique morphological characteristic and distribution pattern of C thyrocytes that can be easily recognized microscopically; however, the total numbers of C thyrocyte profiles and their localization within the thyroid lobe are comparable. The study not only broadens the current knowledge of C thyrocytes but can also be valuable for future studies on this subject in both physiological and pathological conditions.This study provides the first detailed comparison of C thyrocyte morphology, distribution within the thyroid gland parenchyma, and position in relation to thyroid follicles between four domestic animals. Some previously suggested features have been confirmed, complemented, and clarified, and some unique features have been reported for the first time. Our results indicate that the studied species of


To experimentally evaluate the effects of preoperative fasting duration on distant organ liver in renal ischaemia-reperfusion (IR) injury.This is an experimental study. In the study, 3 groups were formed. In Group A, abdominal laparotomy was carried out after 12 hours of preoperative fasting without any IR damage. In Group B, IR injury was carried out after 12 hours of preoperative fasting, and abdominal laparotomy was carried out, in Group C after 2 hours of fasting after IR injury. Apoptosis, congestion, balloon degeneration, nuclear pleomorphism, and leukocyte infiltration were examined histopathologically and tumor necrosis factor-alpha (TNF-α), interleukin (IL) -1 beta, IL-6, and IL-10 were evaluated biochemically.p=0.020) with significantly lower levels determined in Group C than in Groups A and B (p=0.021). Similar rates of mild nuclear polymorphism were seen with no statistically significant difference determined between the groups (p>0.167). A statistically significant difference was determined between the groups in respect of the congestion scores (p<0.001), with a lower score in Group C than in Groups A and B, where the scores were similar .A statistically significant difference was determined between the groups in respect of postoperative IL-10 levels (With this result, it would be correct to say that the short preoperative fasting period has protective effects on the liver tissue. Renal ischaemia reperfusion (IR) damage can occur for many reasons, but usually due to shock, sepsis, organ transplantation, and vascular surgery. High mortality rates can be seen with this condition linked to distant organ failure such as the heart, lungs, brain, and liver. The liver and the kidneys are organs that work jointly in the regulation of the homeostatic mechanisms of the body, and the metabolism and excretion of drugs and toxic products. There are studies in the literature that have defined a close cross-linked relationship between the kidneys and the liver in IR models. The destructive effects of renal IR damage on liver tissue and function have been shown to be formed through reducing antioxidant capacity by triggering oxidative stress and inflammation. Together with the inflammatory process started by hypoxia and IR damage, several inflammatory molecules start to be released from neutrophils, lymphocytes, macrophages, and primarily endothelial cells. The important inflammatory molecules released are tumour necrosis factor alpha and beta , some interleukins , and adhesion molecules.The distant liver effect asssociated with renal IR continues to be a subject of research. Although the pathogenesis of distant organ damage is thought to be multifactorial, the formation of reactive oxygen species (ROS) is the mechanism usually held responsible. The response of the body to insulin starts to decrease, accompanied by glucogenolysis, gluconeogenesis, and fatty acid oxidisation. At this stage, insulin resistance halts the cellular intake of glucose and the glucogen reserves in the liver starts to empty. Hyperglycemia is seen in the circulation associated with insulin resistance. Immune, endothelial, and neural cells are affected by this condition, organ damage and infections occur, primarily in the kidneys and liver, and glycolysis is stimulated. Consequently, more free radicals are formed inflammatory markers rise.Preoperative fasting of patients causes several perioperative metabolic changes. Studies have shown that a prolonged fasting period increases insulin resistance, the inflammatory response to surgery, and oxidative stress. Studies have shown that reducing the preoperative fasting period, providing sufficient hydration, and the consumption of carbohydrate-rich drinks within this program are safe.Enhanced Recovery After Surgery (ERAS) protocols have gained popularity in recent years. This is an evidence-based, multidisciplinary treatment method encompassing the perioperative period, which aims to minimize organ dysfunction and accelerate patient recovery and discharge.The preoperative administration of carbohydrate also causes a lesser acute inflammatory response. Studies have demonstrated lower C-reactive protein (CRP) and IL-6 levels in patients given preoperative carbohydrates compared to patients with a long preoperative fasting period.In renal IR models in the literature, there is no clear proven information related to the effects of the preoperative fasting period on distant organ liver tissue. The aim of this study was to evaluate the effects of the preoperative fasting period on the liver as a distant organ in an experimental model of renal IR. Tissue pathologies were examined with the biochemical parameters of TNF-α, IL-1β, IL-6, and IL-10, which are markers of increased oxidative stress and the release of free radical oxygen molecules in the liver following IR.Approval for the study was granted by the Animal Research Ethics Committee of Health Sciences University Ankara Training and Research Hospital, Ankara, Turkey . This experimental study was started in 2021 after the approval of the ethics committee and the study was carried out in the Animal Laboratory at Ankara Training and Research Hospital, Ankara, Turkey. The study sample comprised 21 male Wistar albino rats, each aging 8-9 weeks and weighting 250±25 grams. An environment was established before the experiment of room temperature 22-25°C, a 12-hour light/dark cycle , and humidity in the range of 55-60%. The rats had free access to standard rat food  and tap water. The 21 rats were separated into 3 groups of 7. The anaesthesia procedure at the start of the study was applied to all the rats intraperitoneally with 90 mg/kg ketamine  and xylazine 10 mg/kg intraperitoneal . A blood sample was obtained from all the rats to determine the preoperative biochemical basal values. Laparotomy was applied to all the rats with a midline incision of approximately 3 cm.The National Institutes of Health Guiding Principles has been taken as a guide regarding the care and use of animals used in this experimental study.Sham group: following a preoperative 12-hour complete fasting period, abdominal laparotomy was carried out, and liver tissue was removed without the creation of renal ischaemia. Liver tissue was removed following reperfusion.Following a preoperative 12-hour complete fasting period, abdominal laparotomy was carried out. The internal organs were retracted and then the left kidney pedicle was clamped. Ischaemia was formed for 20 minutes, then the clamp was opened and a 20-minute period of reperfusion was provided. Liver tissue was removed following reperfusion.Following a preoperative 2-hour complete fasting period, abdominal laparotomy was carried out. The internal organs were retracted and then the left kidney pedicle was clamped. Ischaemia was formed for 20 minutes, then the clamp was opened and a 20-minute period of reperfusion was provided.The liver tissues obtained were placed in dishes containing 10% formaldehyde solution, which had been previously labelled for histopathological examination. After removal of the liver tissue, a second blood sample was obtained from the vena cava inferior of each rat for biochemical examination of the postoperative values. All the rats were then sacrificed with cervical dislocation.The liver tissues of the rats were fixed in 10% formaldehyde solution for 12-36 hours. The samples were separated into pieces macroscopically and one piece was sampled from each sample. Following routine de-paraffinisation with xylene and graded alcohol solutions, the sections were embedded in paraffin and 4 µm slices were cut. These slices were applied with a hematoxylin and eosin (H&E) staining protocol, and then evaluated under an Olympus BX51 microscope for apoptosis, congestion, balloon degeneration, nuclear pleomorphism, and leukocyte infiltration. Scoring was applied as follows: I) for congestion, 0=none, 1=mild (<50% vascular), and 2=significant (>50% vascular); II) for balloon degeneration, 0=none, 1=mild , and 2=widespread (at least one cell in a magnification area x100); and III) for nuclear pleomorphism, 0=present and 1=absent.The TNF-α, IL-1β, IL-6, and IL-10 levels in the serum samples were measured with the solid phase sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) method using the TNF-α ELISA Kit , IL-1β and IL-10 ELISA Kit , and the IL-6 ELISA Kit  according to the manufacturer’s instructions.p<0.05 was accepted as statistically significant.Data obtained in this study were analyzed statistically using the Statistical Package for the Social Sciences, version 17.0  software. Conformity of continuous variables to normal distribution was assessed with the Shapiro-Wilk test, and homogeneity of variance with the Levene test. Descriptive statistics were stated as median (25th-75th percentiles) for continuous variables and ranked variables were also shown as numbers and percentages. The presence of a significant difference in the biochemical measurements from preoperative to postoperative within the groups was examined with the Wilcoxon signed rank test. The significance of differences between groups in respect of biochemical measurements and histopathological scores were evaluated with the Kruskal-Wallis test. When the results of the Kruskal-Wallis test were found to be significant, the Dunn-Bonferroni test was applied to determine the group causing the difference. Correlations between biochemical measurements and between these and histopathological markers were examined using Spearman’s rank number correlation analysis. In all the potentially multiple comparisons, Bonferroni correction was applied to check type 1 error. A value of p=0.028). The preoperative and postoperative differences were similar for all 3 markers.According to Bonferroni correction, there was no statistically significant difference between the groups in respect to preoperative and postoperative IL-1β, TNF-α, and IL-6 levels (p=0.205).The preoperative IL-10 levels were seen to be lower in Group C than in the other 2 groups, but the difference between the groups was not statistically significant (p=0.020) with significantly lower levels determined in Group C than in Groups A and B. Statistically significant p-value between groups was in Group A-C (p=0.020) and Group B-C .According to the results obtained, there was a correlation between IL-1β and TNF-α in respect to the preoperative, postoperative, and change from preoperative to postoperative levels. When the IL-10 levels increased postoperatively compared to the preoperative level, the IL-1β and TNF-α levels also increased . A statistically significant difference was determined between the groups in respect of the congestion scores (p<0.001), with a lower score in Group C than in Groups A and B, where the scores were similar . No statistically significant difference determined between the groups in respect to vacuolar degeneration (p>0.167) although there was a greater number of rats in Group B with significant vacuolar degeneration (The results of the effects of renal IR were examined with pathological examination of the liver tissues to determine histopathological changes. Accordingly, apoptosis, congestion, leukocyte infiltration, cytoplasmic vacuolar degeneration, and the formation of nuclear polymorphism were examined . Similarneration .In this study, the effects of renal IR on liver damage were evaluated with biochemical and histopathological parameters in a rat model. Following the 20-minute reperfusion period after ischemia, both IL10 level and liver congestion and vacuolisation were seen to be less in the group with a shorter fasting period due to less inflammation and organ damage. 20 minutes slight damage with ischemia, 22 minutes moderate damage with ischemia, and 24 minutes histopathologically proved that there is ischemia and severe tubular necrotic damage. In our study, we used the warm IR model in accordance with the literature, we applied ischemia for 20 minutes to allow mild damage instead of severe necrotic damage in order to compare the effects of a condition on IR damage rather than administering a drug or substance.Hesketh et al’s it was reported that following IR or bilateral nephrectomy, kidney damage alone and independently of the etiology could cause liver damage. Kelar et al evaluated the extra-renal effects of acute kidney damage and it was reported that increasing levels of TNF-α and growth factor expressed from hepatocytes had an effect.The effect on the liver in the early stages of kidney damage after IR is thought to start with an increase in oxidative stress with proinflammatory cytokines, a decrease in anti-oxidants, and the formation of apoptosis. In an experimental study by Golab et al, applied renal ischaemia for 30, 45, and 60 minutes followed by a one hour of reperfusion in a rat model, and in contrast to the current study, found the TNF-α levels to be high in all 3 groups. It can be considered that if the reperfusion period in the current study had been longer, different values could have been determined. In addition, in the histopathological evaluation of the study by Hussein et al, congestion and hydropic degeneration were determined in the 45-minute group and leukocyte infiltration in the 60-minute group. No leukocyte infiltration was determined in any group in the current study, less congestion was observed in Group C, applied with the shorter fasting period, and numerically more vacuolar degeneration was seen in Group B.Hussein et al 60 minutes of reperfusion was applied after 3 different periods of renal ischaemia of 30, 45, and 60 minutes. Liver functions and the effects on tissue were examined. When the IL-10 and TNF-α levels were evaluated in the liver tissue, the greatest damage was determined in the 60-minute ischaemia group. In our study, we found that the level of IL-10, an anti-inflammatory mediator, was lower in the 2-hour fasting group, due to less inflammation caused by the short fasting period.In another study by Mehri et al, However, the subject of preoperative fasting remains a matter of debate. Although nil by mouth from midnight before an operation, reduces the risk of pulmonary aspiration, other benefits are not known. A long period of fasting causes an increase in insulin resistance and catabolic pathways, and this condition can trigger postoperative complications.Enhanced Recovery After Surgery is defined as the development of multidisciplinary perioperative protocols, including many applications to improve the patient outcomes. The aim of ERAS is to reduce surgical stress. A reduced preoperative fasting period has been proven to have several positive effects for the patient after surgery, such as shorter length of stay in hospital, reduced insulin resistance, and thereby more rapid wound healing and fewer postoperative complications. In patients with a short fasting period, peripheral glucose uptake and oxidisation in the early period are reduced.While preoperative fasting can activate endogenous resistance to stress, the preoperative consumption of carbohydrates can improve subjective well-being measurements of the patient, such as thirst, hunger, irritability, and headache associated with preoperative fasting. investigated the effects of IR damage with preoperative nutrition periods and reported that a short fasting period reduced IR damage and protected organ function. In the current study, the effects on liver tissue of IR damage were evaluated with different fasting periods. Better scores were obtained in the histopathological examinations in the group with a short fasting period.Van Hoorn et alExamination of a limited number of parameters due to inadequate laboratory conditions.The problem of extrapolating pharmacological data from preclinical to clinical must not be neglected.In conclusion, in our study, it was observed that short preoperative fasting period in renal IR injury caused less difference in both inflammatory parameters and liver histopathology compared to long fasting period. From these results, it would be correct to say that a short preoperative fasting period has protective effects on liver tissue, as a distant organ.


Endurance and resistance exercises, alone or in combination, induce metabolic changes that affect tryptophan (Trp) catabolism. The kynurenine pathway (KP) is the main route of Trp degradation, and it is modulated by the inflammatory and redox environments. Previous studies have shown that KP metabolites work as myokines that mediate the positive systemic effects related to exercise. However, it is poorly understood how different exercise modalities and intensities impact the KP. The aim of this study was to characterize the effect of two different exercise modalities, military diving and swimming, on the KP and the redox environment. A total of 34 healthy men from the Mexican Navy were included in the study, 20 divers and 14 swimmers, who started and stayed in military training consistently during the six months of the study; 12 Mexican men without fitness training were used as the control group. Physical fitness was determined at the beginning and after 6 months of training; criteria included body composition; serum levels of Trp, kynurenine (KYN), kynurenic acid (KYNA) and 3-hydroxykynurenine (3-HK); the glutathione ratio (GSH/GSSG); and malondialdehyde (MDA).. Results showed a significant loss of body fat in both the diver and swimmer groups. Compared with the control group, divers showed a decrease in Trp and 3-HK levels, but no changes were observed in the KYN/Trp, KYNA/Trp or 3-HK/Trp ratios, while swimmers showed a decrease in KYN levels and an increase in the KYNA and 3-HK levels. Additionally, divers showed a decrease in the GSH/GSSG ratio and an increase in MDA levels, in contrast to the swimmers, who showed a decrease in MDA levels and an increase in GSH/GSSG levels. Our findings suggest a differential shift in the KP and redox environment induced by diving and swimming. Swimming promotes an antioxidant environment and a peripheral overactivation of the KP. Exercise is widely recognized to improve whole-body performance, and it is one of the most powerful modulators of human metabolism in health and disease ,2,3. AerTrp catabolism through KP produces several metabolites with redox, immunomodulatory and neuroactive properties ,17,18. TThirty-four healthy men from the Mexican Navy were included in this study: 20 divers and 14 surface and helicopter rescue swimmers (SHRS) from the School of Search and Rescue and Diving  of the Mexican Navy with previously inconsistent exercise. Physical fitness criteria were determined at the beginning  and end of the training programs (after 6 months), evaluating cardiovascular risk, cardiorespiratory fitness (CRF), metabolic equivalents (METs), as well as BC calculating body fat percentage (BFP) and muscle mass percentage (MMP) by anthropometric measurements. We used a sedentary control group of 12 Mexican men without previous training or exercise records.This study was approved by the Research and Ethics Committees of the Instituto Nacional de Rehabilitación “Luis Guillermo Ibarra” (CONBIOETICA-09-CEI-03120171207). All naval personnel were informed of the benefits and risks of the investigation prior to signing an institutional informed consent approval document to participation in the study.The physical and tactical military training performed was that described in the surface rescue swimmer and diving and underwater works of the ESBUSREB manual of the Mexican Navy. Training was progressive in terms of weight increase, repetitions to successfully completing the sets of each exercise and maximal repetitions. The 6-month training program is described briefly.Dry-land training. This training was the same for divers and rescue swimmers; the training consisted of 20 min sessions two times per week with an intensity of 60–80% of maximum heart rate. This dry-land training consisted of combined anaerobic RE and aerobic EE designed to work on strengthening the upper trunk and core muscles to improve muscular strength and power, as well as neuromuscular development, particularly in the arms and trunk. EE exercises were used to increase CRF. The training consisted of warm-up, resistance force exercises , plyometrics, power exercises , Fartlek training and functional exercises.Tactical divers’ training. Divers’ physical training program highlighted RE exercises with weight training (necessary to maintain muscle mass and an acceptable fitness level). RE exercises consisted of two-hour immersion at a 1–5-m depth in the pool, 3 times per week, and 1.5-h immersion at a 60-m depth in the sea, 3 to 4 times per week, over 6 months whilst breathing compressed air (21% oxygen and 79% nitrogen) in an open circuit system and wearing neoprene suits  in sea diving. This training included apneas at a low depth and horizontal apneas of 25 m to learn to control breathing, stay calm and purge the viewfinder under water. The diving activities consisted of rescue diving, revision of axles, propellers, discharges and suctions of ships.Tactical surface and helicopter rescue swimmers’ training. SHRS were equipped with the mental capacity, swimming technique, efficiency, flexibility, strength and endurance to work for 30 min in heavy seas. Tactical training consisted of EE conditioning through crawl swimming with equipment , and body towing (800 m/28 min) to learn how to approach, carry and release survivors; 4 apneas, which consisted of 25 m in length at 40 s intervals, were induced 3 times per week.Cardiovascular risk. The stratification of the cardiovascular risk was determined before starting the military training program (beginning) through a clinical evaluation, including spirometry and electrocardiogram at rest.Cardiorespiratory fitness. CRF was evaluated using the Course–Navette test, which measures maximal aerobic power and indirectly the maximum oxygen uptake (METs) [e (METs) . This teBody Composition. BC was assessed at the beginning and at the end of training program by anthropometric measurements through three indices: (a) body mass index (BMI); (b) BFP and MMP, and (c) waist circumference (WC). The weight, height, diameters, circumference and 25-skinfolds were obtained according to the International Society for the Advancement of Kinanthropometry (ISAK) guidelines [idelines . To avoiAnthropometric measurements. Prior to the anthropometric measurements, naval personnel were asked not to have carried out any type of exercise and to undergo a 12-h fasting, wearing only shorts without lotions, oils or body creams. A calibrated skinfold caliper (Slim Guide) and a short-branch anthropometer (Ces-corf) were used to measure skinfold thicknesses. The measurements were made by two certified level 2 anthropometrists.BMI. This index was calculated by dividing weight (in kg) by height (in square meters) (kg/m2). Height and weight were obtained using a portable stadiometer  and a calibrated digital flat scale (Omron Brand model Hbf-514 C), respectively.Muscle mass and body fat percentage. MMP and BFP were calculated using the Drinkwater and Ross equation [equation ,33 and tequation . The refequation . The refequation  and BFP WC. WC was the screening tool used to estimate weight status in relation to potential disease risk. This tool was measured using a measuring tape (Rosscrafty Lufkin) according to the ISAK protocol for the diagnosis of metabolic diseases in men. A WC ≤ 90 cm is considered as healthy while > 90 cm as a risk [s a risk .3-HK, KYN, KYNA, Trp, glutathione reduced form (GSH), oxidized glutathione (GSSG), N-ethylmaleimide (NEM), O-phthaldialdehyde (OPA), thiobarbituric acid (TBA) and trichloroacetic acid (TCA) were obtained from Sigma-Aldrich . All other chemicals were of the highest commercially available purity. Solutions were prepared using deionized water.Blood samples. Blood samples were collected to determine the serum levels of malondialdehyde , glutathione levels and KP metabolites. Two peripheral blood samples were taken at different times from each participant. The first one was taken prior to starting a military training program. The second one was taken 6 months later once the training was finished. For both samples (beginning and 6 moths), the participants were asked not to have carried out any type of exercise and to undergo a 12-h fasting. Blood samples were collected by venipuncture, in a BD vacutainer serum tube ; serum was immediately separated by centrifugation, then samples were passed through a 0.2-µm filter. Later, samples were separated in two tubes: (1) the first one for immediate determination of the redox state (GSH/GSSH ratio and MDA) and (2) the second was stored at −80 °C until analysis for kynurenines determination.w/v)), mixed immediately and placed on ice to confer stability for GSH quantification. Then, samples were centrifuged at 14,000× g for 20 min at 4 °C. The supernatant was used for GSH (5 µL) and for GSSG (30 µL) determination. For GSSG determination, NEM and dithionite were added, then the samples were treated with OPA to obtain the isoindole. Fluorescence was determined at 370 nm of excitation and 420 nm of emission . The concentrations of GSH and GSSG were obtained by interpolation in the standard curve. The results are expressed as µmol/L.An adapted fluorometric method was used to evaluate GSH and GSSG ,39. In bg for 10 min. Later, the optical density of the supernatant was determined at a wavelength of 532 nm. Results were expressed as a micromolar of MDA (mol/L).Lipoperoxidation was evaluated through the production of thiobarbituric acid reactive species (TBA-RS) . Serum sg and 4 °C. The supernatant was used for kynurenines determination [Serum samples were treated with 200 µL of perchloric acid (6%) and vigorously mixed. Then samples were centrifuged at 14,000× mination ,42,43,44The 3-HK levels were measured using a reverse HPLC method with electrochemical detection. The sample (40 μL) was injected onto an Adsorbosphere Catecholamine C18 reverse phase column  at a constant flow rate of 0.6 mL/min with a mobile phase containing 9% triethylamine, 0.59% phosphoric acid, 0.27 mM EDTA and 8.9 mM heptane sulfonic acid. The 3-HK retention time was ~11 min.Serum neopterin was measured using a commercially available competitive ELISA kit  according to the manufacturer’s protocol. This immunoassay had an intra- and inter-assay coefficient of variance <8% and <10%, respectively, and a sensitivity of 0.094 ng/mL.p < 0.05. All statistics were calculated with Graph Prism 9.1.0. .The changes in BC, physical fitness, Trp catabolism and neopterin levels at the beginning and end of training were assessed using the Wilcoxon signed-rank test. Comparisons between groups were performed using the Kruskal–Wallis test with Dunn’s test for multiple pairwise comparisons. Spearman correlation was used to assess the association between variables. Statistical significance was set at A total of 46 males were included in this study. The mean age of the control group  was 23.1 ± 0.66, of the diver group was 27.2 ± 0.86 and of the SHRS group was 28.6 ± 1.14 years . The thr2), with an improved BC by decreasing BFP and increasing MMP, without changes in METs or redox markers. Additionally, this group showed an increase in 3-HK serum levels and a decrease in neopterin levels (an inflammation marker) (2) despite decreasing BFP and improved CRF (METs ˃ 12). Additionally, this group showed a decrease in Trp serum levels, neopterin and the GSH/GSSG ratio, and an increase in the Trp/3-HK ratio  . The SHRHK ratio .The serum KP metabolites and redox environment induced in the diver and SHRS groups at the end of training (6 months) were compared to the serum levels of the control group . The divWhen the ratios were calculated, the interpretation was similar to that obtained with the serum metabolites levels, since the diver group did not show a difference compared to the control group. The SHRS group maintained a higher ratio of KYNA/Trp and 3-HK/Trp, confirming the over activation of KP in this group .The redox environment was evaluated through the levels of the main antioxidant (GSH) and MDA . The divPairwise correlations among BMI, BFP, Trp, KYN, KYNA and the 3-HK/Trp ratio are shown in In this study, we explored the KP metabolites and redox environment in the serum of Mexican naval personnel to investigate the changes associated with swimming and diving, in combination with a chronic EE and RE as part of a military training program. Our results showed the differences in the Trp catabolism and redox environment of divers and SHRSs over the 6 months of training. After six months of consistent exercise, both groups showed improved BC by losing body fat, but only divers showed increased MMP. The SHRS group lost weight, and despite decreasing BMI, they remained in the overweight category, but with good BC, and showed improved cardiorespiratory fitness (METs ˃ 12); additionally, this group showed decreased circulating Trp levels and an increased 3-HK/Trp ratio, suggesting an overactivation of the KP, leading to de novo NAD+ production. In addition, when the glutathione ratio was determined in both groups after six months of training, only the SHRS group showed increased levels of this antioxidant compared with the levels observed at the beginning of training, while neopterin was decreased.The negative correlation of BMI with KYNA, KYN and Trp levels, as well as of BFP with KYNA and Trp, and the positive correlation of BMI with the 3-HK/Trp ratio in SHRSs suggest that improving SHRSs’ BC by losing body fat but maintaining adequate muscle mass could influence Trp catabolism throughout the increment in free Trp in the blood due to lipolysis during exercise, leading to an increase in free fatty acids, which bind to albumin, releasing Trp into the blood . It coulTo evidence the differential metabolism of Trp, the redox environment and the long-term adaptation effect between diving and swimming, we decided to add a sedentary control group. The results of our study showed that despite having the same average EE and RE in the dry-land training, the metabolism shifted between divers and swimmers. The diver group presented reduced Trp and 3-HK circulating levels when compared to the control group; however, there were no changes in KYN/Trp, KYNA/Trp or 3-HK/Trp ratios, indicating that the Trp catabolism through KP is not modified by diving after 6 months of training. In addition, the diver group presented an oxidant environment when compared to the control group since the GSH/GSSG ratio decreased and the MDA levels increased . These cInterestingly, unlike the diver group, the SHRS group showed an increase in Trp catabolism, confirmed by the decrease in KYN circulating levels, as well as by the increment in KYNA and 3-HK circulating levels, and confirmed by the increase in KYNA/Trp and 3-HK/Trp ratios vs. the control group. The increase in circulating KP metabolites could be associated with the higher oxygen consumption and the requirement of energy during SHRSs’ tactical training, in which EE predomination led to an overactivation of KP, providing the cofactor NAD+ de novo to be used in the energetic metabolism activated by the EE ,54,55. MShifts in KP metabolism induced after a 12-week exercise program  have been observed in healthy older men over 65 years of age. In this case, all the isoforms of KAT increased in skeletal muscle in healthy older men, suggesting exercise as a potential KP modulator for aging to improve the declines in mood and cognition induced by age ,58. AnotOur study has several limitations. First, the sample size was small in both study groups. Second, no major changes were observed in the 6-month evaluation period since both groups had practiced swimming and diving once before entering the ESBUSREB, allowing their bodies to adapt to their respective modality of exercise. Third, we had no control over the diets of the study groups; therefore, we do not know the content and the availability of the macronutrients that may have influenced Trp catabolism. Fourth, the degree of stress, depression and anxiety was not evaluated in this population; however, we are considering these evaluations together with CRF—which has been related to improve memory performance and as-sociated with lower depressive symptoms ,72 in fuHerein, the Trp catabolism and redox status adaptation induced by two exercise modalities, swimming and diving, was characterized. Swimming induced a shift in circulating Trp metabolites and improved the antioxidant environment compared to a sedentary population. These findings pave the way for new studies in which swimming at different intensities could be studied as a potential therapeutic adjuvant in many diseases presenting fluctuations in brain KP metabolites with neuroactive properties.


The growing availability of biologics, which until now have only been available for T2-high asthma, has been further enriched by the arrival of tezepelumab. (2) Methods: This review aims to evaluate the baseline characteristics of patients enrolled in RCTs of biologics for severe asthma to understand how they could potentially predict outcomes and how they can help differentiate between available options. (3) Results: The studies reviewed demonstrated that all biologic agents are effective in improving asthma control, especially with regard to reducing exacerbation rates and OCS use. As we have seen, in this regard, there are few data on omalizumab and none yet on tezepelumab. In analysing exacerbations and average doses of OCSs, pivotal studies on benralizumab have enrolled more seriously ill patients. Secondary outcomes, such as improvement in lung function and quality of life, showed better results—especially for dupilumab and tezepelumab. (4) Conclusion: Biologics are all effective, albeit with important differences. What fundamentally guides the choice is the patient’s clinical history, the endotype represented by biomarkers , and comorbidities . Asthma is a disease characterised by a great heterogeneity of immunological pathways and clinical manifestations, and it requires an approach based on personalised medicine and the study of phenotypes and endotypes ,2. Over This review aims to evaluate the baseline characteristics of the patients enrolled in RCTs of biologics for severe asthma, to better define the current landscape and understand how they could potentially predict outcomes, how they can help differentiate between available options, and to give useful insights to clinicians for a more appropriate choice.1), exacerbations, OCSs, and symptoms, which can quantify the response to biologics [The choice of biologics is crucial in the management of patients with severe uncontrolled asthma. In the literature, the presence of a mixed phenotype varies by 37–47%, depending on the case ,9. This iologics . These pFive biologics are currently available for the treatment of severe allergic , eosinophilic , and mixed asthma with atopic–eosinophilic phenotype (dupilumab), along with tezepelumab . In the 1 of 40–80%), and a recent clinically significant history of exacerbations despite high doses of ICSs, long-acting beta-agonists (LABAs), and other control agents. The patients had received either omalizumab or a placebo for 28 weeks. During this treatment period, the rate of clinically significant exacerbations was 26% less in the active treatment arm compared with the placebo group (p = 0.0002). Severe asthma exacerbations and emergency department visits were significantly reduced, and asthma symptoms and morning peak expiratory flow were significantly improved. EXCELS was a post-marketing observational cohort study undertaken by the Food and Drug Administration (FDA) to evaluate the long-term safety of omalizumab [p < 0.001), severe exacerbations (p = 0.023), hospitalisations (p = 0.003), and overall scores on the asthma control questionnaire (ACQ) (p < 0.001). Finally, 62.7% of patients in the active treatment group had reduced or stopped using OCSs, compared with 30.4% in the control arm. The role of combined biomarkers as possible predictors of treatment response was explored in the EXTRA study, which included 850 patients with severe allergic asthma [Omalizumab is a recombinant humanised mAb IgG1 anti-IgE that binds to circulating IgE, preventing its binding to the receptors of mast cells and basophils, and blocking the release of histamine and other mediators ,16 Figu. It is ualizumab . This stalizumab . Patientc asthma . OutcomeMepolizumab is an anti-IL-5 IgG1k mAb approved for severe eosinophilic asthma (SEA) . Numeroup = 0.003), and benralizumab Q8W resulted in a 70% lower annual exacerbation rate than placebo (p < 0.001). At the end of the study, no significant effect on FEV1 was found. A post hoc analysis evaluated the rate of patients in clinical remission among pooled patients in the SIROCCO/CALIMA or ZONDA trials [Benralizumab is an IgG1 mAb targeting the α subunit of the IL-5 receptor. It activates an antibody-mediated cellular cytotoxicity mechanism, leading to a profound depletion of eosinophils and their precursors in the blood and tissues  Figure . In the A trials . In the A trials . MoreoveA trials .1 and asthma control, especially in patients with higher T2 biomarkers (BEC ≥ 300 cells/µL and FeNO ≥ 25 ppb) [1. Specifically, the mean daily dose of prednisone was reduced by 70%, compared with 42% in the placebo group [1, asthma control, and HRQoL, regardless of the presence or absence of atopy [Dupilumab is a fully human IgG4 mAb directed to the alpha subunit of the shared receptors of IL-4 and IL-13 (IL-4/13Rα), simultaneously blocking the IL-4/IL-13 signalling pathway ,46 Figu. The saf 25 ppb)  . Among the patients without type 2 inflammation (baseline BEC < 150 cells/μL and baseline FeNO < 25 ppb) enrolled in the NAVIGATOR study, the therapeutic effect of tezepelumab was positive, albeit with values generally lower than those in patients with type 2 biomarkers. However, the effect was considered clinically significant. Unfortunately, in the phase 3 RCT SOURCE, with 150 patients randomised into 210 mg tezepelumab Q4W or placebo for 48 weeks of treatment, the primary endpoint of the OCS-sparing effect compared with the control group was not met [1 provocative dosage (PD15) with tezepelumab was 1.9 (95% CI 1.2–2.5) versus 1.0 (95% CI 0.3–1.6) by doubling the doses with placebo (p = 0.06). Nine (45%) subjects in the tezepelumab group and three (16%) in the placebo group had a negative PD15 test at week 12 (p = 0.04). The airway tissue eosinophil and bronchoalveolar lavage levels decreased by 74% and 75%, respectively, in the tezepelumab arm, compared with a 28% increase and 7% decrease in the placebo arm (p = 0.004 and p = 0.01), respectively. This study led to an editorial in which it was noted that an even greater effect could have been achieved if only patients with severe mannitol AHR were recruited [1 and patient-reported outcomes (PROs) and reduced type 2 biomarkers compared with placebo in all patients, as well as in those with severe allergic asthma. DESTINATION is an ongoing phase 3 RCT involving patients who completed the NAVIGATOR and SOURCE studies [The phase 3 programme called PATHFINDER brought interesting results, but also some queries. The NAVIGATOR trial randomised 1061 patients  Table 3Table 3.  not met . A stati not met . We can  not met . In thisecruited . In addi studies . The priThe almost absolute majority of patients enrolled in severe asthma RTCs are primarily classified as having T2-high endotypes . At the With regard to blood eosinophilia, it is interesting to note how the baseline values differed between pivotal studies, due to the selection of patients based on partially different characteristics and who were at least theoretically more suited to the mechanism of action of the various biological agents . The eosIt should be noted that the baseline level of BEC affects important outcomes, such as exacerbation for all mAbs examined, even in anti-IgE, which does not target these cells but can also have indirect effects on eosinophils ,26,64 T. Higher Regarding atopy, all patients had this characteristic in the case of anti-IgE , along w1 improved with higher baseline FeNO values and increased with higher baseline FeNO . These results were independent of BEC, confirming that FeNO was the main biomarker for the management of patients treated with dupilumab [FeNO is a useful biomarker for T2-high asthma phenotyping and a predictor of the response to some biologics. Its production is induced by pro-inflammatory cytokines, including tumour necrosis factor-α, IL-1b, IL-4, and IL-13 . The omaupilumab . Concernupilumab . Therefoupilumab .1 and ICS/LABA doses were similar [Examining the baseline patient characteristics of patients enrolled in clinical trials, they differed—at least in part—not only with regard to biomarkers, but also in the level of disease severity. This resulted in different exacerbation rates and prednisone doses, while FEV similar . Focusin similar , at base similar , 3.8 in  similar , 2.0 in  similar , and an  similar . Thus, tp = 0.002), in which 62.7% reduced or were weaned off OCSs. In the MENSA study, the mean dose of OCSs was 12.6 mg, while the mean dose of patients enrolled in SIROCCO was higher (15.2 mg). No data were available for the LIBERTY ASTHMA QUEST and NAVIGATOR studies because their endpoints were completely delegated to OCS-sparing RCTs. In the dupilumab LIBERTY ASTHMA VENTURE and tezepelumab SOURCE studies, the mean baseline doses were 11 mg and 11.8 mg, respectively [In analysing steroid use, as pivotal omalizumab clinical trials were conducted many years ago, no specific OCS assessment was performed. For this reason, an average baseline dose of prednisone was not reported in INNOVATE. An OCS-sparing study (EXALT) was subsequently conducted . Eighty-ectively ,57. Whenectively ,41, althectively . The mea1 reported in the INNOVATE study was predicted to be 61% . At the end of the treatment period, an improvement of 190 mL was obtained. In the MENSA study, the baseline FEV1 value was 1.73 L, and an improvement of 98 mL was reported at the end of the study. The SIROCCO study had a baseline value of 1.65 L, with an improvement of 159 mL. The QUEST trial reported an FEV1 value of 1.70 L, with an improvement of 340 mL after treatment. The NAVIGATOR study had a baseline FEV1 of 1.8 L, with an improvement of 230 mL.In all investigations, respiratory function was considered as a secondary outcome. Nevertheless, it was still interesting to evaluate the overall behaviour of the biologics analysed. The baseline FEV1, confirming the effectiveness of these agents in this respect.These results show that the efficacy of mAbs differs in the improvement of respiratory function. Dupilumab was the biologic that obtained the best data, followed by tezepelumab  . In genep < 0.001 vs. placebo). In addition, dupilumab improved lung function, asthma control, and HRQoL by suppressing type 2 biomarkers compared with placebo in both subgroups. Benralizumab did not satisfy the outcomes in the pivotal phase 3 OSTRO study in patients with CRSwNP, because it failed to improve the score of the Sinonasal-22 (SNOT-22) outcome test during the first surgery, and the use of OCSs was not statistically significant among the treatment groups [1 + 320 mL in the NP group and + 160 mL in the overall population). This study confirmed the efficacy of anti-IL-5Rα in patients with NP but, as there were no statistically significant improvements in the SNOT-22 scores and the other outcomes described, the FDA requested additional data. For this purpose, a second phase 3 trial called ORCHID  is underway, the main endpoints of which are nasal polyp burden and patient-reported nasal blockage [Comorbid conditions in severe asthma are common. They complicate management and may affect patient outcomes by contributing to poor disease control. For this reason, identifying comorbidities is important. Comorbidities are numerous, can be pulmonary or extrapulmonary, and are more or less frequently associated with asthma. Among the most common are gastroesophageal reflux disease, bronchiectasis, obesity, allergic rhinitis, depression, diabetes mellitus, and cardiovascular diseases. The most evaluated comorbidity in clinical trials was chronic rhinosinusitis with CRSwNP . All of t groups . Howevert groups . The rect groups . The subblockage . Prelimiblockage . A subseblockage . To confblockage . Currentblockage —a phase 1. Carefully defining the real efficacy of the different drugs and their correct positioning can also be very useful in the case of overlaps, which can potentially make a patient eligible to receive more than one mAb. In this regard, identifying the predominant mechanism, key biomarkers, and comorbidities is the winning strategy. A history of atopy with sensitisation to perennial aeroallergens and early-onset asthma may predict response to omalizumab. For this purpose, the total IgE level is a biomarker that is useful only for calculating the personalised dose, but not for predicting the response [Over the last 15 years, we have seen a radical change in the management of severe asthma due to the arrival of more options represented by biologics. These biologics have all been focused on the treatment of the T2-high endotype until the advent of tezepelumab, which addresses a large unmet need in patients with T2-low asthma. Fortunately, all randomised controlled trials demonstrated that all mAbs are effective in improving asthma control, especially with regard to reducing the exacerbation rate and the use of OCSs. As we have seen, in this regard, for various reasons, there are few data on omalizumab—and none yet on tezepelumab, as the SOURCE study missed its primary endpoint. Fortunately, a new trial is ongoing that could resolve this important shortcoming . Howeverresponse . With reresponse ,65. The response ,40,88. Fresponse ,48. Thisresponse . This coresponse .1 compared to omalizumab and mepolizumab. Not only is it difficult to compare the efficacy of mAbs, the very development of these agents has also always been very complex, as demonstrated by the numerous biologics that have failed during RCTs [Due to the lack of head-to-head studies, a few meta-analyses have been conducted in recent years to compare the efficacy of biologics. Without forgetting the limitations of this type of study, generally only slight differences in efficacy and safety have emerged between the various agents ,96. A veing RCTs .A fundamental aspect in considering the process of choosing the correct therapeutic option is the type of comorbidity present, which is closely associated with the type of inflammatory process. Atopy and allergic rhinitis guide the choice and efficacy of omalizumab . ConversDespite the great advances that have been made in the knowledge of molecular mechanisms, many questions about severe asthma remain open and unanswered, especially regarding the profound understanding of the inflammatory microenvironment and its contributions to clinical expression. Asthma is an increasingly common disease, and severe refractory asthma in particular has a high impact worldwide. For this reason it is necessary to further improve its management . In the


Leptospira. Leptospirosis is transmitted mainly through contact with contaminated rivers, lakes, or animals carrying Leptospira. Human leptospirosis has a wide range of non-specific clinical manifestations ranging from fever, hypotension, and myalgia to multi-organ dysfunction, which severely hampers the timely clinical diagnosis and treatment of leptospirosis. Therefore, there is an urgent clinical need for an efficient strategy/method that can be used for the accurate diagnosis of leptospirosis, especially in critically ill patients. Here, we report a case of a 75-year-old male patient with clinical presentation of fever, cough, and diarrhea. Initial laboratory tests and a computed tomography (CT) scan of the chest suggested only tuberculosis. The patient was finally diagnosed with pulmonary tuberculosis (PTB) combined with leptospirosis by sputum Xpert MTB RIF, epidemiological investigations, and delayed serological testing. Furthermore, through metagenomic next-generation sequencing (mNGS) of clinical samples of cerebrospinal fluid (CSF), urine, plasma and sputum, the causative pathogens were identified as Mycobacterium tuberculosis complex and Leptospira spp. With specific treatment for both leptospirosis and tuberculosis, and associated supportive care , the patient showed a good prognosis. This case report suggests that mNGS can generate a useful complement to conventional pathogenic diagnostic methods through more detailed etiological screening .Leptospirosis is a zoonotic infection caused by the pathogenic  Leptospira infection  to the second stage of severe multisystem damage    with potailures) . Metagenailures) . Here, tA 75-year-old male patient from Yongjia county  visited our hospital  on 16 November 2021 (defined as day 0) with the manifestation of fever accompanied by expectoration for 6 days and diarrhea for 5 days.quaque octa hora, q8h)], levofloxacin , and fluid infusion.The patient had visited a local clinic and received oral medication (the detail was unknown) 6 days ago due to fever and paroxysmal cough with yellow sticky phlegm. The patient had a maximum body temperature of 39.0°C and felt no chills then . One day later, the patient started to have an episode of watery diarrhea with a stool frequency of four to five times per day, which is accompanied by pains in bilateral thighs and shanks. He then visited the Second Affiliated Hospital of Wenzhou Medical University on the same day , where he was administered with piperacillin sodium and tazobactam sodium  on day 2 to enhance the anti-inflammatory effect and reduce pulmonary exudation. Continuous renal replacement therapy (CRRT) was simultaneously applied. On day 3, the patient produced blood-tinged sputum. Xpert MTB RIF using sputum identified the presence of rifampicin-sensitive Mycobacterium tuberculosis complex (MTBC) (data not shown). In association with the chest CT result  every morning ], isoniazid tablets , ethambutol tablet , and pyrazinamide tablets . After CRRT, the level of serum creatinine decreased during day 2 and day 3. However, the patient became restless and delirious at the night of day 3. Since he was unable to communicate with, tolerate being in bed, or cooperate to the treatment, CRRT was forced to be terminated and sedation was applied.Anti-infectious treatment was empirically administered with sulperazone  and moxifloxacin  on day 1, combined with supportive care for relieving cough, reducing sputum, and regulating intestinal flora and fluid infusion. The patient still manifested with persistent fever, chest distress, and tachypnea. Oxygen was then supplied at a rate of 6 L/min using an oxygen mask to maintain an oxygen saturation of 95%. The patient had less urine and progressively elevated creatinine. He was then given methylprednisolone [40 mg, I.V. infusion 2O. CSF examination was as follows: WBC, 2 × 106 cells/L; protein, 0.30 g/L; chloride, 127 mmol/L; and glucose, 5.2 mmol/L. There was neither Cryptococcus neoformans nor MTBC (via Xpert MTB RIF) detected. The samples of CSF, urine, and plasma prepared on day 4 were subjected to mNGS, by which the DNA sequences of L. interrogans (without MTBC) were detected with read numbers of 4, 9, and 2, respectively  kit by the Wenzhou Center for Disease Control and Prevention (reported on day 4 after samples being sent for mNGS). Therefore, the diagnosis of leptospirosis was verified. In combination with the heightened inflammation in bilateral lungs shown by the chest CT screen on day 4 (L. interrogans) were detected with a read number of 4 , DNA extraction and library construction (PCR-free) using an NGS Automatic Library Preparation System  (Homo sapiens sequences in NCBI nucleotide (nt) databases (for eliminating human sequences) using bowtie2 v2.3.5.1  using BLASTN v2.10.1+  , NGS on v2.3.5.1 , and a mv2.3.5.1  and bowtv2.3.5.1 . The micv2.10.1+  for furtLeptospira. Animals infected with Leptospira, such as rats, pigs, and cattle, can excrete the bacillus in their urine and contaminate water and soil over a long period of time  in southern China  and had an onset and admission in November, information that is consistent with the region/seasonal pattern of leptospirosis prevalence . In termg region , and focvia genus-specific IgG-ELISA and PTB via Xpert MTB RIF, and further complemented with mNGS, which detected the species-level etiology of leptospirosis , besides detecting MTBC and excluding other potential etiologies. The species-level clarification of leptospirosis enables the retrospective epidemic investigation of the genus Leptospira, which contains multiple pathogenic species , while in less frequent circumstances, it could also affects other organs   . Here, in sputum Table 2.Leptospira stimulate monocytes and macrophages, which then express more responsive inflammatory cytokines   . Leptospd IL-10) . Thereforognosis . ContinuIn conclusion, (1) the outbreak of leptospirosis has significant regional and seasonal preferences, and clinicians should pay close attention to patients’ epidemiological history and be alert to the possibility of leptospirosis; (2) China is a high-burden country for TB; therefore, once cases that could not be explained by a single pathogen/disease are encountered, comorbidity should be taken into consideration to reduce the possibility of misdiagnosis and missed diagnosis; (3) mNGS, as a novel detection method, could assist the diagnosis of leptospirosis timely; and (4) continuous hemodialysis is beneficial to the prognosis of patients suffering from leptospirosis with renal failure complicated with tuberculosis, in addition to treatment for both leptospirosis and tuberculosis.The datasets presented in this study can be found in NCBI with the SRA accession PRJNA827932.The studies involving human participants were reviewed and approved by the board of Ethics at Wenzhou Central Hospital (WCH)/The Dingli Clinical College of Wenzhou Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.JS and XJ are the primary physicians who provided diagnosis and treatment of the patients. JS, WW, KW, and RR collected and analyzed clinical and sequencing data. CN, GH, SZ, FC, and YY prepared some paragraphs of the manuscript in Chinese. JS, WW, KW, and RR wrote the manuscript. All authors have read and approved the final version of the manuscript.Authors WW, KW, YY, and RR are employed by MatriDx Biotechnology Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.