Corona Virus Disease 2019 (COVID-19) is currently prevalent in most countries around the world. It has become a common threat to global human health because there is no specific cure and no targeted treatment for this disease at this stage. Xuanfei Baidu granule (XFBD) included the traditional Chinese medicine prescription in COVID-19 diagnosis and treatment Plan  released in August 2020, which has played a great role in the diagnosis and treatment of COVID-19 in Wuhan, China. This paper intends to evaluate the efficacy and safety of Xuanfei Baidu granule in the treatment of COVID-19.The search strategies of different websites were searched on Cochrane Central controlled Trials Registry, PubMed, excerpt database, Web of science, China National knowledge Infrastructure, Chinese Science and Technology Journal Database, WanFang and other websites. All qualified studies were confirmed to include randomized controlled trials. The search time range was from January 1, 2019 to February 28, 2021. In the meanwhile, the list of references and related reviews was checked. Two evaluators were responsible for the extraction and management of the data independently. The literature quality was evaluated according to Cochrane manual 4.2.2. Heterogeneity test and Meta analysis were carried out by Review Manager V.5.3 software. The bias risk included in the study was evaluated by Cochrane “bias risk” tool, and the relevant statistical data were evaluated by GRADE3.6 evidence quality grading system.This study intends to evaluate the efficacy and safety of XFBD in the treatment of COVID-19 from 4 aspects, including nucleic acid negative conversion time, average hospital stay, clinical symptom improvement rate and lung computed tomography improvement rate.The conclusion of this scheme intends to provide evidence for judging whether the intervention of XFBD on COVID-19 patients is effective or not.CRD42021245640 On March 11, 2020, the World Health Organization declared COVID-19 a global pandemic. Since then, it has become a major global public health problem in the 21st century. Up to March 29, 2021, SARS-CoV-2 has caused 127832853 infections and 2797203 deaths, and the cumulative number of confirmed cases and deaths is still on the rise according to the Johns Hopkins Coronavirus Resource Center. COVID-19 is caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). The early symptoms of COVID-19 include fever, cough and fatigue. Some patients are mainly characterized by gastrointestinal symptoms, such as vomiting and diarrhea. A week later, the patients developed chest tightness, dyspnea and respiratory distress, and some patients rapidly developed into acute respiratory distress syndrome ((ARDS)) and septic shock, and even died. The late stage of COVID-19 can cause serious damage to a variety of organ functions of patients. According to a retrospective study of COVID-19 in critically ill patients, 67.3% of patients with ARDS,28.9% had acute renal injury, heart injury accounted for 23.1%, and abnormal liver function accounted for 28.9%. In the meanwhile, as a sudden and pandemic infectious disease, COVID-19 causes extreme fear and uncertain emotional reactions in the public. It often leads to negative social behavior, which may involve anxiety, depression, insomnia, aggression, depression and other public mental health problems. As a consequence, how to effectively control the rapid spread of COVID-19 and how to effectively treat the rising number of diagnosed patients has become an urgent worldwide problem to be solved.The epidemic of Corona Virus Disease 2019 (COVID-19) appeared in Wuhan, China at the end of December 2019, The prescription, with Ma Xing Shigan decoction as the core, is composed of raw ephedra 6 g, bitter almond 15 g, raw gypsum 30 g, raw almond 30 g, raw plaster 30 g, Atractylodes macrocephala 10 g, patchouli 15 g, artemisia grass 12 g, Polygonum cuspidatum 20 g, verbena 30 g, dried Reed root 30 g, Fructus thunbergii 15 g, tangerine red 15 g, raw licorice 10 g. Adding Atractylodes macrocephala and patchouli to help remove dampness, Polygonum cuspidatum clearing heat and detoxification, verbena activating blood circulation and dredging collaterals are used together to play the effect of dispelling lung dampness, clearing heat and penetrating evil, purging lung and detoxification, XFBD has played a significant effect in the clinical treatment of the first line of epidemic prevention and control in Wuhan. In the meanwhile, pharmacological studies on XFBD show that XFBD has a regulatory effect on inflammatory factors such as IL6, chemokine CXCL8, and related T cells . It contributes to inhibit the storm of inflammatory factors and overactivated immune response caused by 2019-nCoV infection. It is crucial for the early regulation of inflammation induced by virus infection. XFBD's proprietary Chinese medicine preparations have been approved for sale in China in March 2021.Xuanfei Baidu granule (XFBD) is one of the 3 preferred prescriptions in the “COVID-19 diagnosis and treatment Plan (eighth Edition)” issued by the National Health Commission of China. It was jointly formulated by Zhang Boli, academician of the Chinese Academy of Engineering, and Professor Liu Qingquan, dean of Beijing traditional Chinese Medicine Hospital affiliated to Capital Medical University, in the first line of anti-epidemic, for the treatment of mild and ordinary COVID-19 patients.XFBD combined with conventional antiviral drugs in the treatment of COVID-19 has been widely used in clinic. However, it is lack of systematic evaluation and other evidence-based medical evidence to support because most of the current studies on XFBD are the latest single center, small sample clinical randomized controlled trials. As a consequence, this scheme through the collation of literature for systematic evaluation and meta-analysis, to evaluate the efficacy and safety of XFBD in the treatment of COVID-19 patients, providing more comprehensive evidence for the large-scale clinical application of XFBD.22.12.1.1Randomized controlled trials.2.1.2The subjects were diagnosed or suspected patients with COVID-19, and their diagnostic criteria met the definition of “COVID-19 diagnosis and treatment Plan (eighth Edition)”. The diagnostic criteria are as follows:2.1.2.1There is any 1 item in the history of epidemiology and conforms to any 2 items of the clinical manifestations according to the comprehensive analysis of the following epidemiological history and clinical manifestations; If there is no clear history of epidemiology, it conforms to any 2 items of the clinical manifestations, and novel coronavirus's specific IgM antibody is positive, or 3 items of the clinical manifestations.2.1.2.1.1The travel or residence history of the reported community within 14 days before the onset of the disease; Have a history of contact with novel coronavirus infected patients or asymptomatic infected persons within 14 days before the onset of the disease; Have contact with patients with fever or respiratory symptoms from the case reporting community within 14 days before the onset of the disease; Cluster onset .2.1.2.1.2COVID-19-related clinical manifestations such as fever and/or respiratory symptoms; The imaging features of COVID-19 mentioned above; The total leukocyte count was normal or decreased and the lymphocyte count was normal or decreased in the early stage of the disease.2.1.2.2Patients who meet the conditions of a suspected case and have one of the following etiological or serological evidence:Novel coronavirus nucleic acid positive was detected by real-time fluorescence RT-PCR; Viral gene sequencing showed high homology with the known novel coronavirus; Novel coronavirus specific IgM antibody and IgG antibody were positive; Novel coronavirus specific IgG antibody changed from negative to positive, or the titer of IgG antibody in convalescent stage was 4 times or more higher than that in acute stage.2.1.3XFBD+ routine antiviral drugs were used in the test group and conventional antiviral drugs were used in the control group.2.1.4Effectiveness indicators: nucleic acid test negative rate, lung computed tomography results improvement rate, symptom improvement rate , the incidence of adverse reactions.2.2All uncontrolled trials and non-randomized controlled trials; historical controls (comparison of the results of studies conducted in 2 different periods); Comparison between disease and non-disease groups; Trials distributed according to patient characteristics ; The control group was treated with other clinical trials of non-western medicine, such as other types of traditional Chinese medicine or proprietary Chinese medicine; Clinical trials with irregular evaluation indexes or without detailed publication of treatment results, clinical trials without statistical basic data; Reviews, animal experiments, special reports of adverse reactions and non-clinical efficacy studies such as pharmacology and pharmacokinetics; Obvious errors or defects in trial design.2.3The data were collected from the clinical research literature on the treatment of COVID-19 with XFBD published in biomedical journals from 1919 to 2021. PubMed, CochraneLibrary, Web of Science, excerpt database, China National knowledge Infrastructure, Chinese Science and Technology Journal Database, WanFang, etc. Were selected to screen, the WHO International Clinical Laboratory trial Registration platform  and clinical trials  ongoing research was also searched. References that met the inclusion criteria of the study would be reviewed one by one to avoid omissions.The Chinese database was searched by the combination of title or key words and subject words, and the key words were as follows: The key words in English database were as follows: The subject words combined with free words were used for retrieval, the specific retrieval was as follows Table 2.42.4.1Two researchers independently screened the literature, extracted data and quality evaluation and cross-checked, and in case of differences, solicit the opinions of the third party according to the established screening criteria. First of all, researchers read the title and abstract of the literature for preliminary screening, excluded the literature that obviously did not meet the inclusion criteria, and further read the full text for rescreening to determine whether it was included or not. The literature screening process was given in the (PRISMA) flowchart of “preferred report items for systematic review and meta-analysis” Fig. .2.4.2The researchers used Excel to establish a data extraction table, which included: Basic information of the literature: title, author, journal published, year; Basic situation of study subjects, grouping, course of disease, baseline, diagnosis and inclusion/exclusion criteria, randomized and blind use; Intervention measures, course of treatment; Elements of risk bias assessment; Outcome indicators: including effectiveness, safety, etc.If complete information is not available, we would contact the author by email. If the complete data are still not available, the literature would be deleted.2.4.3The bias risk assessment tool recommended by Cochrane system evaluator manual 5.3.0 and the improved Jadad score scale were used to evaluate the quality of the study. The bias risk assessment tool recommended by Cochrane system evaluator manual 5.3.0 was used to evaluate the study quality according to 7 aspects: random method, assignment concealment, subject blind method, blind result evaluation method, data integrity, selective report and other bias.2.4.4The collected information was statistically analyzed by RevMan 5.3.0 software. The counting data were analyzed by relative risk or odds ratio. In the analysis of measurement data, if the measured value of the outcome was based on the meandifference, mean difference analysis obtained by the same measurement unit, and if the same outcome was evaluated but measured according to different methods, the standardized mean difference analysis was used. In addition, the composite results were expressed by the effect value and its 95% confidence interval.2.4.5I2: If I2 ≤ 50% and P > .1, statistical homogeneity was considered to be good. Fixed effect model was used to merge. If I2 > 50% or P ≤ .1, the statistical heterogeneity was large, then the source of heterogeneity was further analyzed. After the obvious clinical heterogeneity was excluded, random effect model was used for Meta analysis. When there was obvious clinical heterogeneity, it should be treated by subgroup analysis or sensitivity analysis, or only descriptive analysis.The clinical heterogeneity and statistical heterogeneity between studies were judged. Clinical heterogeneity was judged according to the similarity of research objects, intervention measures, control and outcome indicators between studies; statistical heterogeneity was evaluated by 2.4.6If a certain outcome index was included in more than 10 articles, the funnel chart and Egger test should be used to analyze whether there was publication bias.2.4.7Sensitivity analysis was carried out by using “leave-one-out” to determine whether the results were affected by different analysis methods (random effect model or fixed effect model), and to prove the robustness of the results.2.4.8For the results of this systematic evaluation, the evidence quality evaluation system GRADE was used to evaluate the evidence quality. The evidence quality was divided into high quality, medium quality, low quality and very low quality. In addition, the recommended grade was divided into strong and weak levels.3 and traditional Chinese medicine often has a unique effect on the treatment of “plague”. XFBD has a definite curative effect in the clinical practice of treating COVID-19, especially in reducing the conversion of mild patients to severe diseases. This study provides evidence of evidence-based medicine for the efficacy and safety of XFBD in the treatment of COVID-19. In addition, it provides a new choice for a variety of treatment options for COVID-19.This study intends to evaluate the efficacy and safety of XFBD in the treatment of COVID-19 patients. COVID-19 belongs to the category of “plague” in traditional Chinese medicine,The limitations of the study are as follows:1.The sample size of randomized controlled trials literature on both is small, or the epidemic reasons can not strictly guarantee the test process because the related studies on XFBD and COVID-19 are more preface, which may affect the reliability of the results;2.The research method of this study has some shortcomings, which reduces the quality and authenticity of the test.It is necessary to pay more attention to expand the scope of literature retrieval, include more high-quality literature, pay more attention to the rigor and standardization of test design and test methods, and strive to provide higher quality evidence for the conclusion in the follow-up research.Conceptualization: Jisen Zhao, Yongcheng Liu.Data curation: Jisen Zhao, Dong Guo, Maoxia Fan.Formal analysis: Jisen Zhao, Maoxia Fan.Funding acquisition: Dong Guo.Methodology: Maoxia Fan, Yongcheng Liu.Resources: Yongcheng Liu.Software: Yongcheng Liu.Writing – original draft: Jisen Zhao, Dong Guo, Maoxia Fan, Yongcheng Liu.Writing – review & editing: Jisen Zhao, Dong Guo, Maoxia Fan.


The first case of hyaline vascular type of unicentric Castleman disease (HV-UCD) was reported more than six decades ago. Since patients with HV-UCD are often asymptomatic and this condition is generally discovered incidentally on imaging tests, most of the previous reports were of mediastinal origin detected by chest radiography. In recent years, improved access to imaging modalities has provided new insights in the diagnosis of this condition. In this study, we reviewed the detailed clinical and pathological findings of 38 HV-UCD cases . The most common site involved was the abdominal cavity (34.2%), followed by mediastinum (23.7%) and retroperitoneum (15.8%). In the abdominal cavity, mesenteric origin was the most common. The mean size of masses was 4.8 cm. Pathologically, thick hyalinized collagen fibers surrounding large blood vessels and calcification were observed . Multinucleated giant cells resembling Warthin–Finkeldey cell were also observed in occasional cases (23.7%). This is a unique paper that summarizes detailed clinical and pathological findings of a large series of a rare disease. The clinical information presented in this paper is more plausible than previous views and is useful for accurate diagnosis and understanding of the disease. Castleman disease (CD) is a rare lymphoproliferative disease, consisting of several subtypes. Each subtype of CD has a different etiology, clinical manifestations, and histology. According to the number of enlarged lymph node regions, CD is divided into two major types: unicentric and multicentric. Unicentric CD (UCD) involves only one lymph node area and is often asymptomatic or mildly symptomatic, whereas multicentric CD (MCD) is associated with systemic symptoms and extensive lymph node involvement. UCD is further divided into two subtypes based on their histological characteristics: hyaline vascular (HV) type and plasma cell (PC) type. The HV type is characterized by increased lymphoid follicles with regressed germinal centers, hyalinized vessels, and hypervascularity within the interfollicular area. The PC type is characterized by hyperplastic germinal centers with sheet-like proliferation of plasma cells within the expanded interfollicular area. It has been reported that most cases of UCD are of the HV type 74.4–91.4%) [.4% 1,2,,2,3,4.CD was first reported in 1954 by Castleman et al. as a case of a 40-year-old man with a large mediastinal mass which turned out to be lymph node hyperplasia revealing follicles with hyalinized foci . HV-UCD UCD can occur at any age and occurs equally in men and women, with a mean age at presentation of approximately 33.8–46.0 years ,8,9,10. The study protocol was approved by the Institutional Review Board of Okayama University, Okayama, Japan. We used opt-out to obtain consent for participation in this study. A total of 38 Japanese patients with HV-UCD were retrospectively identified and included in this study. All cases were retrieved from surgical pathology consultation files from the Department of Pathology of Okayama university between the years 2001–2020. In all cases, the lesions were localized in a single region. Immunostaining for CD20, CD3 and Ki-67 was performed in all these patients, and lymphoma was ruled out. Human herpes virus type 8 infectious status was not examined in this study population. None of the patients had autoimmune diseases or used corticosteroids or immunosuppressants. Other diseases with histopathological features overlapping with those of UCD were also excluded according to the guidelines of Frits et al. . All theThe diagnosis of HV-UCD was made in postoperative surgical specimens and diagnosed by several pathologists at the department of pathology case review meeting. The pathological diagnosis of CD was established according to Keller’s criteria . All casThe demographic and clinical characteristics of HV UCD patients are summarized in Of the 38 included patients, 20 were men and 18 were women with a mean age of 42.8 years  at the time of diagnosis. Majority  of the patients were aged <50 years. Comorbidities included hyperlipidemia , asthma , and type 2 diabetes mellitus , as well as a history of malignant or infectious diseases that led to implementation of imaging studies for the diagnosis.Patients who presented with subcutaneous masses or superficial lymph node swellings were aware of the mass or had localized pain, while most patients with lesions involving the deeper sites were asymptomatic. However, a few patients with retroperitoneal or pelvic masses complained of abdominal discomfort or back pain . Notably, in one case, the presence of paraneoplastic pemphigus led to the discovery of a lymph node lesion. The patient had oral erosions for two years prior to the diagnosis of HV-UCD. Subsequently, shortness of breath and coughing appeared. After a thorough examination, the patient was diagnosed with paraneoplastic pemphigus (PNP) and bronchiolitis obliterans (BO). Plasmapheresis and oral corticosteroids in a dose of 0.5 mg/kg were started along with resection of the lesion of HV-UCD, resulting in improvement of mucosal symptoms. The mucosal symptoms relapsed once during the tapering of steroids, but the PNP has been in remission since then with the maintenance dose of oral corticosteroids monotherapy (0.1 mg/kg). The BO has yet to go into remission but is well controlled with oral corticosteroids and has not had rapid exacerbations. The patient is still alive 20 years after the diagnosis of HV-UCD.In 13 patients, lesions were incidentally found during diagnostic investigations for other comorbidities such as malignancy, gynecologic disease, or infection , whereas some cases were discovered as incidental findings during routine checkups .The most common site of involvement was the abdominal cavity , with lesions of mostly mesenteric origin (7/13). This was followed by mediastinum , retroperitoneum , subcutaneous tissues of the back, buttocks, or extremities , axillary lymph nodes , cervical lymph nodes , hilar lymph nodes , and kidney (2.6%). The kidney mass was found within the parenchyma of the lower pole.18F-fluorodeoxyglucose positron emission tomography-computed tomography (18F-FDG PET-CT) results could be obtained for the following seven patients: patient number 2, 7, 8, 25, 27, 28, 37. The mean maximum standardized uptake value (SUV-max) was 4.8 (range 2.5–9.1). In all these patients, the identical lesions that had 18F-FDG accumulation were surgically resected. There were no other sites showing 18F-FDG accumulation other than the resected lesions.Preoperative differential diagnoses besides HV-UCD included gastrointestinal stromal tumor, solitary fibrous tumor, neurogenic tumor, lymphoma, metastatic carcinoma, and sarcoma in the abdominal cavity; paraganglioma, lymphoma, and sarcoma, in the retroperitoneum; thymoma, thymic carcinoma, neurogenic tumor, embryonal carcinoma, teratoma, and lymphoma, in the mediastinum.The mean maximum diameter of the evaluated masses was 4.8 cm (range 1.7–11.0 cm). The mean diameters of the lesions in the abdominal cavity, retroperitoneum, and mediastinum were 4.1 cm (range 2.0–6.5), 5.8 cm (range 3.0–11.0), and 4.8 cm (range 1.7–9.0), respectively. All patients underwent total resection of the lesions, and none of them received chemotherapy or radiotherapy. Clinical follow-up was carried out in 17 cases, and no recurrences were reported. The mean follow-up period was 72.7 months. There were no abnormal hematological findings such as anemia, increased inflammatory markers, or hypergammaglobulinemia in any of the patients.Representative histological findings of the included cases are presented in The degree of hyalinization was variable. Thick hyalinized collagen fibers were seen in the interfollicular area surrounding large blood vessels while broad hyalinized collagen fibers spreading throughout the lesion were also often observed  . This brIn the follicular and interfollicular area, multinucleated giant cells resembling Warthin–Finkeldey cell were observed in occasional cases  . These mIn this study, we present detailed clinical data and pathological findings of 38 histologically well-defined HV-UCD cases. This is a unique study considering the fact that exclusive reports on HV-UCD are scarce in the existing literature and as such detailed evaluation of multiple cases of HV-UCD will add immensely to the current knowledge of this entity.Data on age, sex, and symptoms were similar to those in the previous reports ,7,8,9,10While malignant tumors , benign tumors , and inflammatory diseases (ascending colon diverticulitis and ischemic enteritis) were present in nine patients, the relationship between these diseases and HV-UCD has been unclear. In severe infections and autoimmune diseases, the adjacent lymph nodes may show reactions such as follicular hyperplasia, hypervascularization, and plasma cell proliferation in the enlarged interfollicular area. However, the histological features of HV-UCD, such as hyalinization of the blood vessels and atrophy of the germinal center, are uncommon in these diseases, so histological differentiation is not considered to be a problem. The patients included in this study did not have any underlying disease that could exhibit histology similar to HV-UCD.One of our patients had PNP and BO in association with CD. A previous report has shown that CD is one of the most common causes of tumor-associated pemphigus, along with non-Hodgkin’s lymphoma and other hematologic tumors ,14. BO iIn this study, four cases of subcutaneous masses, which are considered rare, were also included . One of 18F-FDG avid UCD lesions [There have been several reports of  lesions ,27,28,29HV-UCD is preferably diagnosed by excisional biopsy of the enlarged lymph nodes. Although there are several diseases that show CD-like histopathological features such as unusual morphological variants of follicular lymphoma, angioimmunoblastic T-cell lymphoma, these usually present with generalized lymphadenopathy and are rarely confused with UCD .The finding of altered follicular structure with expanded mantle zone, as shown in In this study, multinucleated giant cells resembling Warthin–Finkeldey cells were observed in the germinal center or interfollicular area in nine cases. Although Warthin–Finkeldey cells are commonly seen in Kimura disease, a rare chronic inflammatory disease with unclear etiology, these are not unique to Kimura disease and are also observed in measles and a variety of reactive or neoplastic lymphoid conditions. In the present study, these multinucleated giant cells were positive for the follicular dendritic cell (FDC) markers such as CD21 and CXCL13, and were considered to be identical to those referred to as dysplastic FDCs in previous reports ,36,37,38The etiology of HV-UCD has been unclear since the disease was first reported over 60 years ago. Although the lineage of cells responsible for the pathogenesis of UCD has not been identified, several studies have hypothesized that a neoplastic stromal cell population may be the precursor of UCD ,42,43,44With this latest body of evidence, we have a better understanding of the pathogenesis of HV-UCD. However, owing to the rarity of the disease, it is necessary to accumulate further evidence with a focus on genetic analysis in the future.In conclusion, our study provides up-to-date clinical and pathological findings of patients with HV-UCD. The detailed clinical information presented in this paper is more plausible than previously thought and is useful for accurate diagnosis and understanding of the disease. Comprehensive genetic analysis of histologically well-defined HV-UCD patients is required as a next step in this area of study to clarify the pathogenesis of the disease.


DRESS syndrome is defined as drug-induced hypersensitivity syndrome with rash, eosinophilia, and systemic symptoms. This syndrome is mostly associated with anticonvulsants, antibacterial and anti-inflammatory drugs. DRESS syndrome is a rare disease and is more frequently seen in adults. We present the first case report of DRESS syndrome in an 8-year-old girl, after 3 months of treatment with isoniazid and rifampicin. After discontinuation of drugs and a short course of prednisolone the girl recovered. After 5 years of follow-up, she is healthy and has no complaints but patch tests with isoniazid and rifampicin remain positive. The reported case emphasizes the importance of thorough medical history and including drug reactions in differential diagnosis. Drug Rash with Eosinophilia and Systemic Symptoms (DRESS) syndrome is a severe adverse drug-induced idiosyncratic reaction occurring 2–6 weeks after the beginning of treatment and is characterized by febrile temperature and skin rash accompanied by hematological abnormalities  and internal organ involvement (hepatitis or other). Diagnosis of this disease is made according to RegiSCAR (European Registry of Severe Cutaneous Adverse Reaction) scoring system .In a recent systematic review of DRESS syndrome in a pediatric population, the authors found 30 different drugs associated with this disease , but in DRESS syndrome is an uncommon disease especially in children due to the difficulty of correct diagnosis , 5. HoweWe report, to the best of our knowledge, the first case history of an 8-year-old girl with DRESS syndrome induced by isoniazid and rifampicin.An 8-year-old girl was diagnosed with mediastinal lymph node tuberculosis. She was treated with isoniazid, rifampicin, pyrazinamide, and ethambutol for 2 months (from January 17 to March 17) and then continued only with isoniazid, rifampicin, and pyridoxine. Liver enzymes  were checked every month and were within normal range.Three months after the start of the treatment (April 18), the patient developed fever (38°C), abdominal pain, nausea, vomiting, and on the next day—jaundice and pruritic maculopapular rash on the face, neck, and trunk. On April 22, the patient was hospitalized in Vilnius University Children's Hospital.9/l; 6.5%). Data of laboratory findings are summarized in Physical examination revealed mild fever (37.6°C), generalized maculopapular exanthema with excoriations on the face, neck, and trunk, icteric sclera, and hepatosplenomegaly. The patient suffered from abdominal pain, nausea, and vomiting. Laboratory tests found abnormal liver function , increased total (347.7 μmol/l), direct (241.9 μmol/l) bilirubin, and GGT (207.9 U/l). Blood test indicated mild eosinophilia . Given the association of fever, rash, eosinophilia, and hepatitis, the diagnosis of DRESS syndrome was made and anti-tuberculosis drugs were suspected to be the cause of this severe drug reaction.Drug hypersensitivity and acute hepatitis were suspected, thus the decision was made to discontinue earlier prescribed drugs. The patient was given prednisolone 1 mg/kg daily, clemastine 1 mg two times per day, and ursodeoxycholic acid 250 mg two times per day. On April 25 eosinophilia dramatically increased to 3.21 × 10Oral prednisolone (30 mg/day) was continued for 6 days. Later, the dose was gradually tapered and stopped after 15 days.Fever (37–38°C) lasted for 7 days, rash gradually resolved after 10 days and liver function started to improve . The patTwo years later the patient was patch tested with culprit drugs. Drug patch tests were performed according to the Guidelines of Contact Dermatitis . To avoiThe patch test for the culprit drugs was repeated after 5 years. It was negative after 48 h and 72 h, but after 1 week (168 h) both isoniazid and rifampicin had become positive (2+) .Skin rash  with fever, hypereosinophilia, lymph node enlargement, and the involvement of various organs is characteristic of DRESS syndrome. The liver is the most frequently involved internal organ. Liver lesions  are seen in 84.5% of patients, and kidney, lung, central nervous system, heart, and other organs comprise from 1.4 to 20.9% ' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.VU was responsible for making the diagnosis, case report supervision, and wrote the first draft of the manuscript. KM was responsible for daily patient care, follow-up, and case report supervision. VU, KM, VK, and OR were involved in the conception of the paper and critically reviewing the article. DV took responsibility in writing sections of the manuscript, editing the manuscript, and literature review. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported for publication from Vilnius University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pretreatment with carvedilol partly prevented the CPX-induced increase in urinary bladder and kidney index, and completely protects from CPX-evoked alterations in serum potassium and creatinine level, but did not prevent histological alterations in the urinary bladder and hematuria. However, carvedilol administration resulted in significant restoration of kidney glutathione (GSH) level and a decrease in kidney interleukin 1β (IL-1β) and plasma asymmetric dimethylarginine (ADMA) concentrations. Not only did mesna improve kidney function, but it also completely reversed histological abnormalities in bladders and prevented hematuria. In most cases, no significant interaction of carvedilol with mesna was observed, although the effect of both drugs together was better than mesna given alone regarding plasma ADMA level and kidney IL-1β concentration. In conclusion, carvedilol did not counteract the injury caused in the urinary bladders but restored kidney function, presumably via its antioxidant and anti-inflammatory properties.One of the major side effects of cyclophosphamide (CPX)—an alkylating anticancer drug that is still clinically used—is urotoxicity with hemorrhagic cystitis. The present study was designed to evaluate the ability of carvedilol to protect rats from cyclophosphamide-induced urotoxicity. Rats were injected intraperitoneally ( Many drugs, excreted with the kidneys, are later accumulated in urine in high amounts and may interact with urothelium for a long time, especially in the urinary bladder during the night. Urothelium, lining the urinary tract from the kidney to the urethra, is a high-resistance barrier preventing the diffusion of a variety of substances excreted by kidneys to the tissues. This specific structure is, therefore, exposed to high concentrations of endogenous waste products as well as some xenobiotics, including drugs. Additionally, the urothelium is exposed to mechanical stretch due to the physiology of the urination process . DespiteDespite many new, targeted biologic therapies in the treatment of malignancies or autoimmune diseases , conventThe mechanism of CPX-induced injury in the urinary tract is multifactorial and triggered mainly by the interaction of acrolein, a toxic CPX metabolite, with urothelium that induces the inflammatory response. Up-regulation of nuclear factor kappa B (NF-κB) is important in causing such cytokines to release as tumor necrosis factor-alpha (TNF-α), interleukin 1β (IL-1β), or IL-6. Additionally, the generation of reactive oxygen species (ROS), overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), involvement of bradykinin, substance P, and platelet-activating factor (PAF) is important or suggested in this pathological pathway. This leads to the destruction of the urothelium, erosions, ulcerations, pain, and bladder overactivity with the possible role of activation of alpha-1 adrenergic receptors ,8,9. In For the prevention of CPX-induced hemorrhagic cystitis and reduction in urinary toxicity, bladder irrigation or forced diuresis with prior hydration with or without intravenous administration of mesna (sodium 2-mercapto-ethanesulphonate) are commonly used. The postulated mechanism of mesna, a sulfhydryl compound, is that it binds the methyl group of acrolein which leads to the formation of thioether, which is non-toxic, and does not trigger the inflammatory process ,13. ReceCarvedilol is a highly lipophilic, third-generation nonselective beta-adrenergic antagonist, additionally acting as an antagonist of alpha-1 receptors with a ratio of beta to alpha blockade of about 10:1. It is also one of the beta adrenolytic drugs described as biased agonists promoting β-arrestin-mediated ERK phosphorylation, which finally suggests inhibition of neoplastic transformation and carcinogenesis ,18. It is postulated that carvedilol, and its metabolites, are potent antioxidants and scavengers of ROS ,20. It iMany studies focused on the preventive potential of carvedilol in anthracycline-induced cardiotoxicity in humans and in a variety of in vitro or animal models ,24,25, bBased on these premises, our study aimed to evaluate the potential of carvedilol in acute, cyclophosphamide-induced urinary tract toxicity. After the adaptation period, on day 1 of the main experiment, the mean body weight in all experimental groups was comparable and no significant differences between groups were noticed . For an explanation of the groups’ abbreviations, please see the Materials and Methods section. In all groups receiving cyclophosphamide intraperitoneally in a single dose of 200 mg/kg, a significant decrease in body weight in comparison to the control group was found with the largest body weight loss in the group receiving only CPX. Administration of mesna (200 mg/kg in divided doses), alone or with carvedilol , partly prevented the body weight decrease (significant differences vs. CON and CPX group). The action was the least pronounced in the CPX-C group A. p = NS in both cases). The urinary bladder, kidney, and liver indexes are presented in p < 0.05 in both comparisons). Carvedilol given alone or in combination with mesna fully prevented the increase in the potassium level in serum and the differences between these two groups and the control group were insignificant, but there were significant differences with the CPX group. In the group receiving CPX, the serum creatinine level was the highest and was significantly different from the control group (1.76 times greater than in the CON). All studied substances fully prevented the serum creatinine level increase and the differences between these groups and CON were insignificant, but significantly different from the CPX group. The action of carvedilol, mesna, and the combination of the treatment was comparable, and no significant differences were noticed between these groups . The mean values and SD for both parameters are presented in The highest serum potassium level on day 4 of the study was measured for the CPX group and was significantly different from CON. Administration of mesna alone only partly prevented the increase in serum potassium level . The changes in hematuria in time in all experimental groups are presented in In the microscopic evaluation of urinary bladders, in almost all studied specimens focal or diffuse urothelium reduction were found; therefore, these changes were not involved in the scoring system as they were nonspecific to any of the performed experimental procedure. The most pronounced changes in a form of inflammatory infiltrations, edema, hemorrhage, or erosions were found in the group receiving only CPX. Additionally, ulceration of urothelium was found in one case. Considering the scoring system of histological changes in urinary bladders, the highest score was calculated for the CPX group and was significantly different from the control group. Carvedilol did not prevent histological injury in urinary bladders and the score value in the CPX-C group was not significantly different from the CPX group, but was significantly different from the CON group. Administration of mesna alone or in combination with carvedilol fully prevented the histological injury and reversed the CPX-induced changes. A significant difference between groups receiving mesna was found in comparison to the CPX-C group. The representative images from urinary bladder histological evaluation are presented in p < 0.01 vs. CON). Administration of mesna  partly reversed the pathological changes expressed in scoring (in both cases the differences between mesna-receiving groups to CON and CPX groups were insignificant). Carvedilol alone did not exert any effect on CPX-induced damages . However, these two groups (CPX-C and CPX) differ in such a way that in the group receiving CPX, interstitial hemorrhages were found in half of the specimens, whereas in the CPX-C group, interstitial hemorrhage was noticed only in one case. From all groups receiving studied substances, the least pronounced injuries were described in the group receiving both mesna and carvedilol, where no cases of interstitial hemorrhage were found. The representative images from kidney histological evaluation are presented in The histological changes in the kidney were less specific and in almost all studied specimens parenchymatous offuscation and hyperemia were described, and similarly to the bladder scoring system, these two parameters are not involved as they are not dependent on experimental procedures. The most pronounced pathologies were described in the CPX-receiving group, where dilated urinary spaces, interstitial hemorrhages, and glomerular atrophy were found in almost all examined samples. Considering the scoring system CPX caused significant injury when compared to the control group  and partly reversed the CPX action in the liver (p = NS vs. CON and CPX). In none of the examined tissues was administration of carvedilol able to reverse the increased level of MDA caused by CPX (significant difference compared to the control group). The drug combination (group CPX-MC) only prevented the CPX-induced changes in MDA level in the urinary bladder and liver (p = NS vs. CON and CPX) but exerted no effect in the kidney . The mean values with the corresponding SD for urinary bladder, kidney, and liver MDA levels are presented in After the single intraperitoneal injection of CPX at a dose of 200 mg/kg, the malondialdehyde (MDA) level was significantly increased in all studied tissues . Administration of mesna alone (group CPX-M) completely prevented the CPX-induced increase in MDA level in urinary bladder and kidney (p < 0.001 vs. CON) and both studied substances  reversed this action, increasing the GSH concentrations . It was also noticed that injection of CPX decreased superoxide dismutase (SOD) activity and the value was the lowest in this group from all experimental groups, but the difference between that and the control group was not significant. In the kidney, the tendency to increase SOD activity in comparison to the CPX group was observed in the case of all groups receiving studied compounds, but there were only insignificant differences regarding the CON and the CPX group. The mean values with corresponding SD for kidney GSH levels and SOD activity are presented in CPX caused a significant decrease in glutathione (GSH) concentration in kidney homogenates (p < 0.001), but additionally, significantly decreased the level of this cytokine in kidney homogenates in comparison to the control group, because the IL-1β levels were lowest in groups receiving carvedilol, especially alone . The differences between the CPX-C group and both groups receiving mesna (CPX-M and CPX-MC) were also significant. The mean values with corresponding SD for kidney IL-1β levels are presented in In the kidney, CPX significantly increased the level of IL-1β in comparison to the control group. Administration of mesna alone failed to reverse this action, but administration of carvedilol not only prevented a CPX-induced increase in IL-1β level . Additionally, the ADMA level is the lowest in the CPX-MC group, significantly lower not only compared to the CPX group but also compared to the CPX-M group . No statistically significant differences in liver DDAH activity in comparison to the control group were noticed. However, the highest liver DDAH activity was measured in the CPX-M group, but it was still insignificant. Serum ADMA level and liver DDAH activity are presented in Administration of CPX significantly increases the plasma ADMA level. All studied substances  completely prevented the CPX-induced ADMA increase  of the CPX dose given in three divided doses , but may be increased up to 160% of the CPX dose in four divided doses or even up to 320% of the daily post-transplant cyclophosphamide dose [In clinical practice, mesna and/or hydration with or without diuretics are commonly used to prevent dose-dependent cyclophosphamide-induced urinary toxicity such as micro- and macrohematuria or hemorrhagic cystitis ,14,41, wide dose . Howeveride dose . Therefoide dose , and somide dose . N-acetyide dose , but theide dose ,47 reveaide dose . Due to the very complex mechanism of CPX-induced urinary toxicity, research focuses on substances inhibiting, e.g., the inflammation cascade or overactivation of oxidative stress. Introducing new natural or synthetic compounds to clinical practice takes a long time of preclinical and clinical tests. Searching for new applications among already registered drugs with known toxicity and pharmacokinetic is a promising way to shorten this time . In expeIn the present study, we have chosen a low dose of carvedilol (2 mg/kg) given once daily by gastric tube for 3 days. The low dose was selected based on the earlier published papers confirming that an intraperitoneal dose of 2 mg/kg given in 12 h intervals is effective in the prevention of I/R kidney injury in rats where carvedilol restored antioxidants levels  and decreased MDA level in kidney tissue altered by I/R procedure. Moreover, doses from 1 to 3 mg/kg exerted the least pronounced hemodynamic effect, whereas higher doses did not give additional protection in the I/R model but produced a decrease in systolic blood pressure and heart rate . The samIn our study, the model of a single intraperitoneal injection of 200 mg/kg of CPX was used to induce hemorrhagic cystitis and kidney injury as it is described in some other papers ,67,68,69CPX, in contrast to ifosfamide, is not uptaken by kidney proximal tubules and the nephrotoxic action of CPX is rather due to the generation of ROS and peroxynitrite, activation of inflammatory pathways, cell membrane disruption, mitochondrial dysfunction and DNA or protein adducts formation by acrolein . In the 4) model of liver injury. Despite some reports about the hepatoprotection of carvedilol in other animal models of liver damage, such as CCl4-induced fibrosis [Liver toxicity was not the primary goal of our study, but we found significantly increased liver index in the CPX group, which was reversed only by mesna, but not by carvedilol alone. Cyclophosphamide may cause abnormal hepatic function and very rarely hepatitis and hepatomegaly . A lot ofibrosis  or I/R ifibrosis , our preThe protein arginine methyltransferase (PRMT) enzymes catalyze the formation of ADMA . ADMA isCarvedilol exerts many properties that can be important in protecting against CPX-induced urotoxicity. Despite the complex mechanism, we did not find significant protection against CPX-evoked cystitis or potentiation of mesna action. It cannot be excluded that direct neutralization of acrolein than antioxidant activity of carvedilol is more important for the prevention of cystitis. Despite a detailed review of the literature available to us, we did not find any paper describing the action of carvedilol in CPX-induced cystitis or nephrotoxicity, so it is difficult to compare our results with other studies. Some reports confirm that carvedilol prevents toxicity induced by other anticancer agents, especially in the doxorubicin- or daunorubicin-induced cardiotoxicity ,56, cispsubst. , mesna subst. , carvedilol subst. , 0.9% NaCl solution, ampules 10 mL , pentobarbital sodium 133.3 mg/mL + pentobarbital 26.7 mg/mL, bottles 100 mL .Cyclophosphamide Male Wistar rats with an average weight of 315.67 g ± 17.98 g were purchased from the Animal Research Center at Wroclaw Medical University  and after 2 weeks of adaptation period, they were randomly divided into five experimental groups . Rats were housed two per cage in standard laboratory conditions of 21–23 °C, 12 h:12 h light:dark cycle, free access to standard rodent feed , and water ad libitum. The experiment was approved by the Local Ethics Committee for Animal Experiments in Wrocław (No. 13/2012). i.p.) injection of 200 mg/kg of CPX. Mesna, as a reference treatment, was given i.p. 20 min before, 4 h and 8 h after CPX administration at a total dose of 200 mg/kg divided in 20%, 40%, and 40% doses, respectively, which was equal to 40 mg/kg, 80 mg/kg, and 80 mg/kg for separate injections. CPX and mesna were dissolved in normal saline and administered in a 5 mL/kg volume. Normal saline solution given intraperitoneally in the same volume and schedule served as a control. Carvedilol was given intragastrically (i.g.) by gastric tube  once daily at a dose of 2 mg/kg, dissolved in 4 mL/kg normal saline solution, a day before, on the day, and a day after CPX administration. Animals in the control group were given normal saline solution i.g. at the same volume and schedule. Animals were sacrificed by cervical dislocation in barbiturate anesthesia 48 h after the CPX administration. Before the CPX injection and after 4 h, 8 h, 24 h, and 48 h, urine samples were taken for assessment of hematuria. Blood samples from the tail vein were collected 24 h before CPX injection and 48 h after. Urinary bladders, kidneys, and livers were separated, weighed, and collected for further histological evaluation and preparation of tissue homogenates. The general organization of the experiment and the nomenclature of the experimental groups is summarized in Hemorrhagic cystitis in rats was induced by a single intraperitoneal (Body weight (BW) was checked and recorded (in grams) once daily in the morning from day 1 to day 4 of the study. After the animals were sacrificed, the urinary bladders, kidneys, and livers were collected and weighed. The urinary bladder, kidney, and liver weights were expressed as the percentage of the total body weight  from day 4 and calculated according to the formula:Forty-eight hours after the CPX administration, blood samples were collected from the tail vein for serum potassium and creatinine measurements. The measurements in serum were made after centrifugation in the certified commercial laboratory just after the samples were collected.Urine samples for hematuria evaluation were collected directly on the Siemens Multistix 10SG test  by a gentle massage of suprapubic area 5 min before CPX or normal saline injection, and 4 h, 8 h, 24 h, and 48 h after CPX or normal saline injection. The magnitude of hematuria was assessed semi-quantitatively (from 1+ to 4+) using the scale on the dipstick, modified in this way, so that 4+ were given when macroscopic hematuria was noticed.Part of the urinary bladder, kidney and liver tissues were homogenized on ice, with lysis buffer  and thereafter centrifuged at 14,000 rpm for 25 min at 4 °C using homogenizer Pro250 . MDA, GSH, SOD, and IL-1β were assayed in supernatants. MDA concentrations were assessed in the urinary bladder, kidney, and liver tissues, whereas GSH concentrations and SOD activity were determined in kidney tissue using the kit and following the instructions of the manufacturers on the MARCEL S350 PRO spectrophotometer . MDA and GSH were assayed using BIOXYTECH-MDA-586 kit and BIOXYTECH GSH-400, respectively , and their concentrations were expressed in μM. The activity of SOD was evaluated using the Ransod kit  following the manufacturer’s instruction, and activity was expressed as U/mg of protein. Total protein concentration in supernatants was measured in a certified laboratory using the Dimension RxL-Max . In brief, in an alkaline solution, cuprum interacts with peptide bond in protein; the amount of measured Cu(II) complex is proportional to protein concentration.Interleukin 1β in kidney tissue was assessed by Rat IL-1β ELISA kit  according to the manufacturer’s instructions on Epoch ELISA Reader  and the results were expressed in pg/mL.On the last day of the study, the blood samples from the tail vein were collected and plasma ADMA level was evaluated using E91301Ge ELISA Kit  according to the manufacturer’s instruction and the results were expressed in ng/mL. Plasma ADMA concentrations were assessed on Epoch ELISA Reader .w/v) in 5% acetic acid) mixed with antipyrine/H2SO4 (antipyrine (0.5% w/v) in 50% sulfuric acid) reagent was added. Next, samples were once more incubated at 60 °C for 110 min and later cooled in an ice bath for 10 min. Serial dilutions of L-citrulline were used as standard. L-citrulline formation was measured at 466 nm. Values were subtracted from respective blanks (without ADMA).DDAH activity was determined in liver tissue homogenates using the colorimetric method on spectrophotometer MARCEL S350 PRO  according to the method described earlier  and exprPart of the urinary bladders and one kidney were fixed in 4% buffered formalin and later prepared for histological evaluation by embedding in paraffin, cutting into 4 µm slices, and staining with hematoxylin and eosin. Samples were coded and assessed blindly by an experienced pathologist using a microscope Olympus BX41  with Fujitsu computer system . In the kidney, the following features were determined for the scoring system: the presence of interstitial hemorrhages, glomerular atrophy, and dilation of urinary spaces. For each pathology, 0 points were given if it was absent or 1 point was given if it was present in a studied sample . In samples of urinary bladders, inflammatory infiltration, hyperemia, edema, ulcerations, erosions, and hemorrhages were determined for the scoring system. For each observed pathology, 0 points were given when absent and 1 point was given if present in the evaluated sample . From representative samples, the images are taken in 100× magnification.p < 0.05. Statistical analysis was performed using TIBCO STATISTICA 13.3 PL . To express experimental data on all figures, means ± standard deviations (SD) were used. Statistical analysis of the effects of the substances on the studied parameters was carried out with the analysis of variance (ANOVA). The equality of variances was checked by the Brown–Forsythe test and specific comparisons between studied groups were later performed with Tukey’s post hoc test. In the case of inequality of variance , the Kruskal–Wallis test was used as a post hoc test. Hypotheses were positively verified if Administration of CPX caused many damages to the urinary bladder and kidneys. The best protection against CPX-induced injury was found to be mesna, which improved most of the studied parameters. Carvedilol failed to counteract the injury caused in the urinary bladders, but restored kidney function as expressed in serum potassium and creatinine levels, and exerted antioxidant and anti-inflammatory action. Additionally, carvedilol and mesna alone or given together prevented CPX-induced plasma ADMA elevation. It cannot be excluded that protective action of carvedilol may be better expressed after longer administration, e.g., 21–28 days, similarly to the duration of breaks between cycles of chemotherapy. The results of our study, along with the data published by other researchers, support the hypothesis of possible nephroprotection exerted by the third generation of beta adrenolytic drugs.


This study aimed to compare the prevalence of hypogonadism between male patients with latent autoimmune diabetes (LADA) and type 2 diabetes (T2DM) and investigate the risk factors for hypogonadism in these patients.This cross-sectional study evaluated 367 male patients with LADA (n=73) and T2DM (n=294) who visited the endocrinology department of Shanghai Tenth People’s Hospital between January 2016 and October 2019 for diabetes management. Sex hormones, lipid profiles, sex hormone-binding globulin (SHBG), glycosylated hemoglobin A1c, beta-cell function, uric acid, and osteocalcin were determined in serum samples. Hypogonadism was defined as calculated free testosterone (cFT) less than 220 pmol/L along with the presence of symptoms (positive ADAM score).The rate of hypogonadism in the LADA and T2DM group were 8.2, and 21.7%, respectively (p=0.017). After adjusting possible confounders, the rate of hypogonadism in the LADA group was comparable to those of the T2DM group. Univariate logistic regressions demonstrated that age, BMI, fasting C-peptide, triglycerides, total cholesterol and uric acid were associated with hypogonadism in men with diabetes, BMI, triglycerides and estradiol were independent risk for hypogonadism in men with diabetes.This is the first evidence to explore the rate of hypogonadism in male patients with latent autoimmune diabetes (LADA). In the population requiring admission to a large urban hospital in China, the rate of hypogonadism was comparable to those of the T2DM group after adjusting for possible confounders. BMI, triglycerides and estradiol were independently associated with the presence of HH in male diabetic patients. Latent autoimmune diabetes in adults (LADA) is a highly heterogeneous disease and the most common form of adult-onset autoimmune diabetes. Patients with LADA initially do not require insulin but eventually become insulin dependent after a rapid decline in beta cell mass and function , 2. ManyHypogonadism is characterized by low concentrations of testosterone and is widely associated with insulin resistance, diabetes, obesity, and metabolic syndrome. Increasing evidence has shown that the high prevalence of hypogonadism in male patients with T2DM and testosterone supplement could improve glucose metabolism and insulin sensitivity in those patients –6. ThereAccordingly, this study aimed to compare the occurrence rate of hypogonadism among male patients with diabetes according to its specific type and investigated the risk factors for hypogonadism in men with diabetes, which would help us to explore the association of hypogonadism and the etiology of hyperglycemia.This cross-sectional study was approved by the Institutional Human Subjects Review Board of Shanghai Tenth People’s Hospital. Written informed consent was obtained from all participants. We evaluated male patients with diabetes who visited the Endocrinology Department of Shanghai Tenth People’s Hospital between January 2016 and October 2019 for better control and management of diabetes. Patients with moderate or severe liver and renal dysfunction, with history of congenital/hypogonadotropic hypogonadism, hyperthyroidism, varicocele, epididymitis, testicle injury, abnormal karyotype, or those who received testosterone replacement therapy in the past 6 months were excluded. The cohort involved 367 patients; of them, 73 patients had LADA; and 294 patients, T2DM.The diagnostic criteria for LADA were glutamic acid decarboxylase (GAD) antibody-positivity, initially non–insulin requiring for at least 6 months, and diagnosed over the age of 30 years . Classic2) was adopted to calculate free testosterone concentrations , and hypertension were recorded. Blood samples were obtained from all patients in the morning after at least 10 hours of overnight fasting. Fasting plasma glucose, fasting C-peptide, glycosylated hemoglobin A1c (HbA1c), GAD antibodies, creatinine, uric acid, total calcium, total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol (LDL-c) were measured. A standard meal load test  was performed, followed by examination of plasma glucose and C-peptide in 120 min . The tottrations .The microvascular disease was defined as presence of macroalbuminuria, diabetic neuropathy and diabetic retinopathy. The macrovascular disease was defined as presence of cerebrovascular disease, coronary heart disease, heart failure, or peripheral arterial disease. Plaques were identified by the ultrasonography test, including the common carotid artery, superficial carotid artery, common femoral artery and superficial femoral artery. The above data were collected by reviewing of all patient’s electronic medical records.Continuous data were compared among the two groups using linear regression analyses. Meanwhile, categorical data were compared among the three groups using logistic regression analyses. Multiple logistic regression models were used to determine the effect of diabetes types on the diagnosis of hypogonadism and investigate the independent risk factors of hypogonadism. Variables with P<0.20 in univariate analysis were included in the multiple logistic regression analyses. All statistical analyses were performed using SPSS version 20.0 software. A P value of <0.05 was considered statistically significant.. There were no significant differences in LH, FSH and cFT levels between the two groups. The mean testosterone, E2 and SHBG levels of the LADA group were different from those of the T2DM group.The mean age of the patients with LADA and T2DM was 53.6 ± 13.2 and 51.9 ± 16.3 years, respectively. Considering the BMI was significantly different in two groups, the comparison of clinical features between them was adjusted by BMI. The LADA group had lower SBP than the T2DM group. The mean fasting c-peptide, postprandial c-peptide, uric acid and triglyceride levels in the LADA group were lower than those of the T2DM group. Meanwhile, the HDL-c levels in the LADA group were higher than those of the T2DM group. The mean DBP, total cholesterol, LDL-c, creatinine and osteocalcin levels were similar between the two groups. The frequencies of microvascular disease, macrovascular disease and plaques were comparable between the two groups . To determine the influence of diabetes types along with confounders on the diagnosis of hypogonadism, multiple logistic models were used to compare the rate of hypogonadism between two groups. After adjusting possible confounders (Model 3 and Model 4), the rate of hypogonadism in the LADA group was comparable to those of the T2DM group .The rate of hypogonadism was much lower in the LADA group than the T2DM group . The incidence rates of hypogonadotropic hypogonadism in the LADA and T2DM groups were 6.8%, 17.6%, respectively (P=0.019). Meanwhile, the incidence rates of primary hypogonadism in the LADA and T2DM groups was 1.4% and 4.1%, respectively (P=0.318) . To dete. Moreover, BMI (OR 1.149 [95% CI: 1.033–1.279]), triglycerides (OR 1.167 [95% CI:1.014–1.343]) and estradiol (OR 0.96 [95% CI:0.976–0.995]) were independently associated with the presence of hypogonadism in the multiple logistic regression analyses. There were no association between hypogonadism status and HbA1c, fasting glucose, osteocalcin, microvascular or macrovascular complications of diabetes, and presence of plaques .The risk factors for hypogonadism in male diabetic patients was explored in the overall population. Univariate logistic regressions demonstrated that age, BMI, fasting C-peptide, triglycerides, total cholesterol and uric acid were associated with hypogonadism in men with diabetes . MoreoveIncreasing evidence has shown that hypogonadism was associated with insulin resistance, diabetes, obesity, and metabolic syndrome , 6. MoreIn our study, the mean total testosterone levels in patients with LADA were significantly different from those in patients with T2DM. This was consistent with previous studies that testosterone levels tended to be normal in patients with T1DM but lower in patients with T2DM , 10. GivTo explore the possible underlying mechanism for the phenomena, the risk factors for hypogonadism in male diabetic patients were explored in the overall population. Consistent with previous studies, majority of cases of hypogonadism in the two groups were hypogonadotropic hypogonadism , 26. ThiIt is widely reported that the high expression of aromatase in the adipose tissue, which could increase the conversion of testosterone into estradiol, leading to decreased gonadotropin secretion . Our datWe also observed an association between uric acid and hypogonadism in our study consistent with the findings of other studies that uric acid was negatively correlated with testosterone levels in patients with T2DM , 23. A pIn addition, triglycerides and total cholesterol were risk factors for hypogonadism in male diabetic patient. The results support the findings that men with hypogonadism may have dyslipidemia and testosterone treatment could significantly decrease total cholesterol and triglycerides , 30. PreThis study has some limitations. First, the exclusion of patients with CKD may be a selection bias for this study; Second, the recruited patients were all inpatients with poor blood glucose or metabolic control, which might increase the risk of hypogonadism; Third, few men without diabetes as a proper comparison may bring about a confounding bias; Fourth, the sample size of patient with LADA was not adequate to detect significant differences. Despite these limitations, we believe that our study is valuable because our findings might not only contribute to further understand the effects of testosterone on glucose metabolism, but also help to better understand the effective way to prevention hypogonadism in male patients with diabetes.In a population of requiring admission to a large urban hospital in China, the rate of hypogonadism was comparable to those of the T2DM group after adjusting confounding factors. The results suggested the association between hypogonadism and the etiology of hyperglycemia was limited in this study. BMI, triglycerides and estradiol were independently associated with the presence of HH in male diabetic patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The Institutional Human Subjects Review Board of Shanghai Tenth People’s Hospital. The patients/participants provided their written informed consent to participate in this study.MZ: designed and wrote. MC, RC, and PY: performed and collected. JG, XC, and CS: participated in recruiting the patients. HS and SQ: edited. All authors contributed to the article and approved the submitted version.This study is supported by grants from the National Key R&D Program of China (No.2018YFC1314100) and the National Nature Science Foundation (No.81601269).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: This study aimed to determine the effect of poppy seed flour (PF) on the physicochemical and spectroscopic properties of low-carbohydrate, high-protein, and gluten-free bread. Methods: The changes at the molecular level were assessed in bread using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Bread prepared with buckwheat, flaxseed, and pea protein was enriched with PF at a concentration of 5–15%. Results: The results showed that the pasting parameters of dough supplemented with PF were significantly decreased compared to the control sample. The obtained bread samples were characterized by good quality and had 14.6% of carbohydrate, 16.3% of protein, 10.2% of fiber, and 4.0% of fat, with a caloric value of 177 kcal/100 g. The addition of PF had little influence on crumb mechanical properties. The ATR-FTIR analyses revealed spectral changes in the region related to protein and carbohydrate structures, as well as changes in band intensity characteristic of α-1,4-glycoside and α-1,6-glycoside bonds. The analyses showed that the main starch skeleton remained clearly visible. Conclusions: PF up to 10% can be potentially applied as a functional ingredient in the production of bread based on buckwheat and linseed flour. Such low-carbohydrate bread can be particularly useful to diabetics.  The rise of several civilization diseases necessitates the development of novel functional bread. In recent years, there has been a lot of focus on developing gluten-free bread recipes; researchers are attempting to produce high-protein bread ,2, with Poppy seeds are one of the world’s most important and oldest oil crops. They contain a high amount of K, Ca, and P (V) ions. This distinguishes poppy seeds as a valuable dietary supplement with potential for use in the production of novel food products. Moreover, research has shown that the content of Cu (II), Zn (II), Pb (II), and Hg (II) ions in the poppy seeds does not exceed the maximum allowable concentration. This signifies that poppy seeds are safe for consumption .Although poppy seed is rich in nutrition, studies on it as a food additive are scarce. Only some researchers have attempted using poppy seeds to produce biscuits  or burgePoppy seeds are also used as a topping for bread . In someThus far, changes in the molecular structure of bread caused by supplementation with various additives, including poppy seeds, have not been investigated in any study. It has been indicated that addition of different food supplements, on the one hand, caused changes in the chemical, mechanical, sensory, and physical properties of starch and, on the other hand, increased the scope of its functional applications. However, it must be emphasized that such practices as modification and supplementation in food processing techniques can cause disruption or changes in certain molecular structures due to, for example, interactions between particular ingredients. This, in turn, can have undesirable effects on the physicochemical, mechanical, and organoleptic properties of food . Hence, Starch is composed mainly of two types of α-glucans, namely amylopectin and amylose, which are connected by α-1,4-glycoside or/and α-1,6-glycoside bonds. These glucans are characterized by a crystalline/amorphous structure, with the short-range order particularly corresponding to the double- and single-helical amylose or amylopectin, which is located in amorphous or crystalline lamellar regions, as well as amylose–amylopectin helix complexes . This cl−1 contained sensitive bands related to the conformation of the starch polymer  and can, therefore, be taken into account in the investigation of, among others, crystallite fusion and multistage retrogradation processes [−1 may be considered in the quantitative determination of the crystallinity index, as those bands may be associated with the respective ordered and amorphous structures within the given food material. Literature reports indicate that attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) has been used to assess the most characteristic infrared bands of starch, and the folding of amylose and amylopectin chains. A study assessed the retrogradation of starch using FTIR and concluded that the spectral region of, for example, 800–1100 cmNo study has, so far, analyzed the enrichment of high-protein and low-carbohydrate bread with poppy seed flour (PF). Therefore, the present work analyzed the rheological properties of PF-supplemented dough, as well as the physical and antioxidant properties of high-protein and low-carbohydrate bread enriched with PF. In addition, the effect of supplementation of PF at various amounts on changes in the molecular properties of the obtained product was determined by a spectroscopic analysis of FTIR bands. The findings of this study could contribute to expanding the knowledge on the impact of PF on starch at the molecular level and the resulting conformational changes in the given product.The typical pasting curves of flour samples supplemented with PF, which were generated based on RVA, are shown in On the other hand, the high-protein defatted PF displayed lower swelling ability due to the stronger bonding between the ingredients and, thus, exhibited lower peak viscosity . It shouThe moisture level of the obtained breadcrumbs remained the same at about 55% . A slighThe crumb of the control bread appeared dark brown in color. This could be due to the darker color of flaxseed flour, which resulted from the presence of a pigment in the outer seed coat . The addThe crumb of the bread enriched with PF was characterized by a higher hardness value, but the increasing share of PF had no significant impact on this parameter . RegardiSamples enriched with 5% and 10% of PF were rated the best by evaluators . These wSupplementation with PF changed the chemical composition of low-carbohydrate bread . EnrichmTPC and AA analyses showed that the lowest TPC was found in PF (0.42 ± 0.06 mg GAE/g d.m.) and the highest in the control flour (1.45 ± 0.13 mg GAE/g d.m.). This suggests that addition of PF in the recipe decreased the TPC of bread . IshtiaqEnrichment of low-carbohydrate bread with 10% of PF caused an increase in the content of analyzed amino acids including asparagine, serine, glutamic acid, proline, alanine, leucine, and valine .trans-9,12-octadecadienoic acid in bread samples fortified with 10% of PF , flaxseed flour , PF , and pea protein powder  were purchased from health food stores. The composition of buckwheat flour, flaxseed flour, and PF was, respectively, 13.1%, 40.1%, and 36% of protein (ISO 20483:2006) ; 3.2%, 8The effects of PF on the pasting properties of bread were investigated using an RVA-4500 system , according to the standard method 22–08 , as described by Wójcik et al. . This anThe control bread samples were prepared based on a previous study , with thUsing the millet seed displacement method, the volume of bread loaves was measured , and theThe color of bread crumb was determined in CIE L*a*b* system using a CR30-16 colorimeter  by assuming the following parameters: illuminant D65 and 10° standard observer. In the CIE L*a*b* color system, L* indicates lightness/darkness (from 0 to 100), a* denotes the degree of redness (+)/greenness (−), and b* represents the degree of blueness (−)/yellowness (+). Measurements were done in triplicate.−1. Curves were plotted using the thickness values of bread crumb with testXpert V 7.1 simulation software  of the testing machine. Based on the curves, parameters such as hardness, cohesiveness, springiness, and chewiness [The texture of bread crumb was assessed using a ZWICK Z020/TN2S testing machine  after 24 and 48 h of baking. For the purpose of measurement, the bread sample (20 mm × 20 mm) cut from the central part of the slice (14 mm thickness) was double-compressed to 60% of its thickness, at a speed of 20 mm shewiness  were detThe sensory properties of low-carbohydrate bread were evaluated by 56 panelists . During the evaluation, attributes such as color, taste, odor, texture, and overall acceptability of bread were assessed using a 7-point ranking scale . The poiThe amino acid composition of bread samples was determined by acid protein hydrolysis as described by Davis and Thomas . The conThe fatty acid composition of the low-carbohydrate bread was determined based on their corresponding methyl esters, which were prepared according to the ISO 12966-2:2017-05 method . Analysig, 10 min). The supernatants were collected and used for biochemical analyses by following Folin–Ciocalteu’s method [For determining the TPC and AA of control flour, PF, and enriched bread samples, their methanolic extracts were prepared. Briefly, 0.5 g of each sample was extracted with 5 mL of methanol using the procedure of Romankiewicz et al. . The exts method  with sliThe AA of OH extracts was assessed by determining their radical-scavenging activity against stable DPPH and ABTS radicals as described by Brand-Williams et al.  and Re eThe half-maximal inhibitory concentration (EC50) or the concentration at which the tested compound showed 50% of the maximum inhibition was calculated in fitted models, based on dose-dependent mode of action .−1 at an excellent resolution of 1 cm−1. Measurements were performed at the Laboratory of the Department of Biophysics, University of Life Sciences in Lublin. Before the analysis, each spectrum was processed using Grams AI software . The infrared spectra of selected samples of control bread and PF-enriched bread were obtained using an IRSpirit spectrometer . For the purpose of measurements, an ATR attachment, i.e., a ZnSe crystal with adequate geometry (truncated at 45 o), was used to ensure 20-fold internal reflection of the laser beam. For each sample, a total of 24 scans were registered. After measurement, the software automatically averaged the results. Before and after each measurement, the crystal was carefully cleaned using ultraclean solvents . Exactly 1 h before and continuously during each measurement, a neutral (N2) atmosphere was maintained inside the chamber. All the spectra were registered at room temperature within the range of 450–3800 cmAll statistical analyses were performed using Statistica 12.0 software , and differences were considered significant at a level of α = 0.05. Each parameter was evaluated using a one-way analysis of variance (ANOVA). If ANOVA showed significant differences, the means were compared by Tukey’s range test.The results confirmed that PF can be potentially applied as a functional ingredient in the production of bread based on buckwheat and linseed flour. Such low-carbohydrate bread can be particularly useful to diabetics. However, the concentration of PF added should not exceed 10% because it may cause a bitter aftertaste. The study showed that optimal PF supplementation resulted in breads with good quality parameters. Compared to the control bread, bread enriched with 10% of PF had a higher loaf volume. In addition, PF supplementation did not cause adverse textural changes. Enrichment of bread with PF also increased the content of valuable amino acids including asparagine, serine, glutamic acid, proline, alanine, leucine, and valine as well as the total amount of fatty acids. Moreover, PF-enriched bread had a reduced caloric value.FTIR analysis revealed that the main starch skeleton in the end product (formed by amylase and/or amylopectin) remained visible. However, the addition of PF clearly affected the positions of certain spectral regions as well as their intensity/spectral shift. The changes observed in protein and carbohydrate content in the end product clearly corroborated with the results reported in other studies. Additives can react strongly with starch at the molecular level, leading to the cleavage of hydrogen bonds and consequently alterations in the conformation of starch. They also infiltrate the molecules of the main starch components and, with increasing concentration, can influence their structures, thereby altering them directly or stimulating them to assume more ordered forms. Changes in starch structure can affect the quality, volume, and texture of bread; however, this warrants more research. In the subsequent papers dedicated to this topic, these problems will be explored in detail by extensive spectral analyses.