Salmonella Typhimurium to various antibiotics. S. Typhimurium ATCC 19585 (STWT) was exposed to ciprofloxacin, gentamicin, kanamycin, and tetracycline to induce antibiotic resistance, respectively, assigned as STCIP, STGEN, STKAN, and STTET. The susceptibilities of the antibiotic-induced resistant mutants to cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, polymyxin B, streptomycin, tetracycline, and tobramycin were determined in the absence and presence of CCCP and PAβN. STCIP showed the cross-resistance to tetracycline and collateral sensitivity to gentamicin (1/2 fold) and kanamycin (1/4 fold). STTET was also cross-resistant to ciprofloxacin (128-fold) and collateral sensitive to gentamicin (1/4-fold) and kanamycin (1/8-fold). The cross-resistance and collateral sensitivity of STCIP and STTET were associated with the AcrAB-TolC efflux pump and outer membrane porin proteins (OmpC). This study provides new insight into the collateral sensitivity phenomenon, which can be used for designing effective antibiotic treatment regimens to control antibiotic-resistant bacteria.The objective of this study was to evaluate collateral sensitivity and cross-resistance of antibiotic-induced resistant  Salmonella is a major foodborne pathogen that causes widespread contamination and infections treatment), and the ΔCtcalibrator is the Ct values for the STWT cells normalized to 16S rRNA . The comparative ΔΔCt method was validated by the amplification efficiencies of the respective target genes  and the reference gene (16S rRNA).Total RNAs in STCIP, STGEN, STKAN, and STTET in association with the induction of antibiotic resistance. The most significant finding of this study was the collateral sensitivity of STCIP and STTET to gentamicin and kanamycin. STKAN showed no collateral sensitivity to ciprofloxacin. The efflux pumps, and outer membrane porin proteins were linked to the collateral sensitivity and cross-resistance. These results can be useful for designing effective antibiotic treatments, such as alternating and combination antibiotic treatments for infections caused by antibiotic-resistant bacteria. Further study is needed to investigate whole genome sequencing of the antibiotic-induced resistant mutants to elucidate the exact genetic alteration responsible for increased antibiotic susceptibility. In addition, a gene knockout and complementation study is underway in our lab to validate the mechanisms associated with collateral sensitivity.Collateral sensitivity has important clinical implications and may transform the approach to antibiotic therapy. By strategically combining antibiotics with collateral sensitivity, it is possible to induce the enhanced susceptibility of bacteria to antibiotics, enhancing bacterial eradication and lowering the risk of resistance development. This concept has great potential not only in human medicine but also in veterinary and agricultural settings, where multidrug-resistant pathogens pose a significant threat to animal health and food production. This study describes cross-resistance and collateral sensitivity in ST


Staphylococci are among the list of problematic bacteria contributing to the global antibiotic resistance (ABR) crisis. Their ability to adopt the small colony variant (SCV) phenotype, induced by prolonged antibiotic chemotherapy, complicates staphylococcal infection control options. Novel and alternative approaches are needed to tackle staphylococcal infections and curb ABR. Manuka honey (MH), a non-antibiotic alternative is recognized for its unique antibacterial activity based on its methylglyoxal (MGO) component.Staphylococcus aureus is well-studied for its international ABR burden, limited data exists demonstrating the effects of MH on S. epidermidis and S. lugdunensis whose pathogenic relevance and contribution to ABR is also rising.In this study, MH (MGO 830+) was tested in combination with gentamicin (GEN), rifampicin (RIF), or vancomycin (VA) against staphylococcal wildtype (WT) and SCVs. To our knowledge, there are no current studies in the literature documenting the effects of MH on staphylococcal SCVs. While in vitro, stable SCVs were observed in GEN treatments only. The addition of MH to these GEN-SCV-induction analyses resulted in complete suppression of SCVs (p<0.001) in all three staphylococci, suggesting that MH’s antibacterial properties interfered with GEN’s SCV induction mechanisms. Moreover, the addition of MH to growth cultures of recovered stable SCVs resulted in the inhibition of SCV growth by at least 99%, indicating MH’s ability to prevent subsequent SCV growth. These in vitro analyses demonstrated MH’s broad-spectrum capabilities not only in improving WT staphylococci susceptibility to the three antibiotics, but also mitigated the development and subsequent growth of their SCV phenotypes. MH in combination with antibiotics has the potential to not only resensitize staphylococci to antibiotics and consequently require less antibiotic usage, but in instances where prolonged chemotherapy is employed, the development and growth of SCVs would be hampered, providing a better clinical outcome, all of which mitigate ABR.The three staphylococci were most susceptible to RIF (0.06-0.24 μg/ml), then GEN (0.12-0.49 μg/ml), and lastly VA (0.49-0.96 μg/ml). The MICs of MH were 7%, 7-8%, and 6-7% (w/v), respectively. Fractional inhibitory concentration (FIC) evaluations showed that the combined MH + antibiotic effect was either additive (FICI 1-2), or partially synergistic (FICI >0.5-1). While all three antibiotics induced SCVs  In nosocomial settings, it is especially problematic to resolve, leading to extended hospitalizations, elevated medical costs, and increased mortality rates  that have gained clinical recognition for their association with infections of indwelling medical devices, examples of which include pacemaker infections, prosthetic valve endocarditis, and infections involving vascular grafts  and rendered the infections they cause difficult to treat . Antibioty rates . Methicity rates . Staphylr grafts .ABR is further complicated by the ability of bacteria, like the staphylococci, to form small colony variant (SCV) phenotypes, a unique bacterial sub-population that, in comparison to wildtype (WT) strains, display a range of atypical characteristics that not only hamper their clinical identification but complicate their pathogenesis . Their r2O2)-mediated versus non-peroxide mediated (®), graded to represent antibacterial potency the higher the UMF value is , rifampicin (RIF), and vancomycin (VA) were employed in this study owing to their clinical relevance and increased reports of resistance , S. epidermidis (ATCC®12228™), and S. lugdunensis (ATCC® 43809™) were used in this investigation. Stock samples of pure cultures were regularly maintained on tryptic soy agar (TSA) grown for 24 hrs at 37°C and stored at 4°C. Rapid purity and species identification tests were performed throughout this investigation using a range of standard methods: colony morphology, haemolysis pattern (on sheep blood agar (BA)), Gram stain, and API® Staph (biomérieux). PCR (16s rRNA) was also performed to confirm the species identity of the generated SCV isolates  (ristics) .2.2®, Canada) were prepared by dissolving 20 mg of antibiotic powder in 10 ml of MHB to obtain a final stock solution of 2 mg/ml. Rifampicin (RIF)  preparations were concurrently prepared in 10 ml of methanol (was a better solvent for this antibiotic than MHB)   was used in this study. This was stored in the dark at 4°C from which fresh stock solutions were made for each assay. Stock solution was aseptically prepared by dissolving 10 g of MH in 20 ml prewarmed Mueller-Hinton broth (MHB) to obtain a final stock solution of 50% (w/v). Stock MH was diluted further in MHB to obtain MH concentrations of 5 – 14% (w/v) (with 1% increments). Stock antibiotic preparations of gentamicin (GEN) and vancomycin (VA) (Avantorhan MHB) . Antibiohan MHB) .2.3S. aureus, S. epidermidis, and S. lugdunensis to MH and the three antibiotics were performed in vitro using the broth microdilution technique according to the CLSI and EUCAST guidelines , 100 µl of fresh log phase bacterial suspension adjusted to 103-105 cfu/ml was added to either 100 µl of premade MH or antibiotic at the aforementioned ranges. Absorbance was measured  at 0hrs and 24hrs following incubation at 37°C (shaking was performed prior to OD (580 nm) readings). The MIC of MH and the MIC of antibiotics were recorded as the lowest concentrations without significant change in OD after 24 hrs. MICs were also confirmed visually  after 24 hrs.MIC determinations for WT idelines . In a st2.4S. aureus, S. epidermidis, and S. lugdunensis were investigated using the checkerboard microdilution assay  against WT on assay  with minThe combinatorial effects were reported as synergistic (FICI ≤0.5); partially synergistic (FICI >0.5-1); additive (FICI 1-2); indifferent (FICI 2-4); or antagonistic (FICI >4) .2.5in vitro and in vivo. In this investigation, GEN, RIF, and VA were individually assessed for their in vitro induction of SCV in the three staphylococcal isolates as previously described , GEN was found to be the only antibiotic to consistently induce stable SCVs. Only stable SCV phenotypes were selected for use in subsequent SCV-MH assays. A time-kill kinetic assay was performed to test the ability of MH-GEN combination to suppress the development of staphylococcal SCV via antibiotic induction . In brie2.7S. aureus, 7-11% for S. epidermidis) in a 96-well plate. The absorbance was measured at 1hr intervals for up to 72 hrs. S. lugdunensis SCVs analyses were performed on Mueller-Hinton agar (MHA) plates due to challenges in detecting growth absorbance using microplate spectrometry. MHA plates adjusted to 6-10% (w/v) MH were prepared and inoculated with 100 μl aliquot suspensions of stable SCVs. Growth was visually monitored every 24 hrs for a duration of 72 hrs.In this investigation, stable SCVs were cultured with MH alone to assess its antibacterial activity on the growth propagation of SCVs. 20 stable SCV colonies were suspended into 2.5 ml of MHB, and 100 μl aliquots of this preparation added to 100 μl of MH  with the corresponding controls. Data were analyzed and mean ± SD was calculated. One-way analysis of variance (ANOVA) was employed to assess the mean differences among and between the groups. P-value ≤ 0.05 was considered significant. Dunnett’s test was performed 33.1S. aureus, S. epidermidis, and S. lugdunensis ranged between 0.06-0.97 μg/ml. In general, of the three antibiotics tested, these staphylococci were most susceptible to RIF (0.06-0.24 μg/ml), followed by GEN (0.12-0.49 μg/ml), and lastly VA (0.49-0.96 μg/ml). The MICs of MH for the three staphylococci were 7%, 7-8%, and 6-7% (w/v), respectively , while the MH+GEN combination lowered MIC 2-fold, which resulted in an additive effect (FIC=1.5). For S. epidermidis, the combination of MH +RIF, MH+VA, and MH+GEN all demonstrated additive effects, with FIC of 1.14, 1.5, and 1.13, respectively. The antibiotic MIC values for this species were lowered 4-fold (GEN), 3-fold (RIF) and 2-fold (VA) when MH was added. For S. lugdunensis, MH +GEN combination demonstrated partial synergy (FIC=0.93) with MIC lowered 3-fold, while the effects of MH in combination with RIF or VA were both lowered 2-fold, with FIC indicating an additive effect of 1.5 for both . The ability of MH to inhibit the in vitro development of antibiotic-induced SCVs was also investigated. The addition of MH yielded no observable SCVs (stable or transient) in all three staphylococci during the incubation period.All three antibiotics (only at concentrations higher than MIC) were capable of inducing SCVs (transient or stable SCVs) in all three staphylococci during the 10-day his time 2. GEN w3.4This experiment compared the antibacterial activity of the antibiotic GEN alone, versus the combination of GEN+MH against WT log phase growth. In general, the results of the time-kill investigation showed that over the 10-day incubation period, treatment of all three staphylococci with the combination of GEN+MH resulted in a decline of at least 3 log in comparison with cultures exposed to GEN alone Figure 2S. aureus exposed to GEN+MH (10X MIC +7%) treatments showed a 3 log10 reduction in growth versus cultures of S. aureus exposed to GEN  (S. epidermidis cultures exposed to GEN+MH (5X MIC +8%) showed 100% growth suppression in comparison to an average growth rate of 1715 cfu/ml in the presence of GEN alone  showed a 2 log10 decline in growth compared to GEN only cultures (4826 cfu/ml vs 78 cfu/ml) (Cultures of S. aureus and S. epidermidis) and on MHA (S. lugdunensis). S. aureus and S. epidermidis instances, the addition of MH resulted in significantly longer lag phases for SCV cultures in comparison to their WT counterparts under the same conditions. In addition, the inhibition of SCVs of all three species was observed at MH concentrations higher than WT MIC  to achieve the same inhibitory effects . The identity of these SCV isolates was confirmed as S lugdunensis using PCR.Following the observation that MH could inhibit the development of antibiotic-induced SCVs, this investigation tested whether MH alone could impede further growth of stable SCVs in culture. MH (6- 11% (w/v)) was added to the harvested stable SCVs from the SCV-induction assay and the growth patterns monitored spectrophotometrically in MHB  was investigated as a potential alternative antimicrobial due to the challenges of antibiotic failure in the efficient resolution of staphylococcal infections, including the clearance of their SCVs in the presence of current antibiotic chemotherapeutics alone. RIF, GEN, and VA, routinely used in the clinical treatment of staphylococcal infections were tested in combination with MH on WT and SCVs of  options . The use options . Old antS. aureus primarily), have mainly reported synergistic effects (FICI ≤0.5), with few others reporting partially synergistic and additive effects (FICIs ranging from 0.87-2)  indicated an additive to partial synergistic activity for the three antibiotic + MH combinations tested Table 1. 0.87-2) . The dif 0.87-2) . Interes 0.87-2) .In this study, it was postulated that the additive and partially synergistic results observed come not only from MH’s unique actions but could have also enhanced those exerted by the individual antibiotics. RIF is a potent bactericidal, exerting action through inhibition of bacterial mRNA transcription, but it also displays a heightened penetration capacity . In thisPseudomonas aeruginosa which is necessary in generating energy to drive this bacterium’s proton-dependent drug efflux mechanism. Without a functional energy generator, antibiotic exportation (via efflux pumps) was reduced, which in addition to MH’s action to increase membrane permeability, greatly enhanced antibiotic activity, thereby rendering this crucial resistance mechanism ineffective . We therefore concluded that for complete clearance of both WT and SCVs to be achieved simultaneously using MH, concentrations >3% that of their corresponding WT MH MIC would produce the best results. We postulate that SCV’s altered metabolism, exhibited by slower growth, a characteristic known to impede antibiotics that depend on WT metabolism, may also hamper other antimicrobials. Therefore, to achieve adequate SCV clearance, both higher and prolonged MH exposure would be the most effective. While it remains unclear why there would be variances in S. lugdunensis detection, this experiment reiterated the importance of using both broth and agar techniques concurrently to confirm antibacterial effects especially when working with SCVs.The extended lag phase of staphylococcal SCVs in comparison to their corresponding WT observed in this study . Nonetheeatments Figure 3Klebsiella pneumoniae in a mouse model. All MRSA-infected mice recuperated after IV treatment with MH, and a substantial reduction of bacterial load was seen in K. pneumoniae subjects , or incorporated into wound dressings, other clinical applications have been documented. A recent study investigated the therapeutic potential of intravenously administered MH to treat septicaemia (caused by MRSA) and New Delhi Metallo-β-lactamase (NDM)-1 producing subjects . Other msubjects , dental subjects , and sinsubjects . To imprry of MH . MH micrry of MH . Sustainin vitro testing demonstrates MH’s efficacy at low concentrations, more in vivo investigations are still needed to determine comparability. Additional clinical data is also required to support the efficacy and usage of MH in infection control.In conclusion, as the current clinical antibiotic arsenal is met with extensive bacterial resistance, and the lack of new and efficacious antibiotic options in the development pipeline, there remains an urgency for concerted efforts to develop novel and alternative measures to circumvent ABR and offer long-lasting clinical interventions. This study alongside others demonstrated the combinatorial effect of MH with three antibiotics, RIF, GEN, and VA in staphylococcal growth control. The addition of MH to antibiotics exerts a two-fold advantage – significantly improved bacterial growth control, and reduction of antibiotic concentrations administered. In addition, with minimal bacterial resistance to MH observed in other investigations, this non-antibiotic alternative stands as a viable alternative particularly in combinatorial therapy of staphylococcal infection, all of which improve the ABR dilemma currently plaguing humanity. These combined findings demonstrate MH’s ability to improve the sensitivity of all three staphylococci to antibiotics by lowering the antibiotic MIC values, which could help augment antibiotic potency. This subsequently would prolong the shelf life of these antibiotics, and in turn alleviate the progression of ABR where staphylococci are involved. With technological advancements steadily growing, there’s potential to expand the therapeutic applications of MH as an alternative treatment in problematic infection control. While The original contributions presented in the study are included in the article/All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


We present a case of a patient with recurrent prostate cancer after treatment for favorable intermediate risk cancer. There was an exceptionally steep increase in PSA from <0.5 to 130ng/mL in 27 months accompanied with the development of bone metastasis. The PSA increase was unexpected. We suspect that this unusual development of metastases must have been caused by an impairment of the immune system caused by his IgG4 disease, and this may have allowed residual prostate cancer cells in the prostate to spread quickly. The influence of IgG4 on cancer is debated. IgG4-RD develops as focal masses and/or diffuse enlargement of target organs, with common phenotypes including autoimmune pancreatitis (AIP), retroperitoneal fibrosis/aortitis and sclerosing sialadenitis/dacryoadenitis and lymphadenopathy.The possibility of an increased malignancy risk in IgG4-RD patients remains controversial. IgG4-related disease (IgG4-RD) is a rare systemic immune-mediated fibro-inflammatory disorder, defined pathologically by a triad of lymphoplasmacytic and IgG4 plasma cell infiltration, storiform fibrosis, and obliterative phlebitis.2The patient was treated at age 70 with brachytherapy for favorable intermediate risk prostate . The brachytherapy implant was inserted with a good quality index (D90 = 192 Gy at day 30). PSA levels declined following the usual rate with a nadir of 0.23 at 24 months post brachytherapy. PSA levels were at 0.29 at 3 years post brachytherapy and some months later top 0.47. Around this time and unrelated to this discrete increase in PSA, the patient was hospitalized with hepatobiliary symptoms. See Concerning his IgG4disease, a scan showed kidney and lung masses, as well as pancreatic masses causing biliary obstruction. A pancreatic biopsy demonstrated fibrosis with lymphoplasmocytic infiltrate and venulitis without thrombosis. The IgG4/IgG positive plasmocyte ratio was normal. IgG4 serum levels were 1,6 g/L (upper limit 1.35 g/L). Therefore, a preliminary diagnosis of IgG4 related-disease was established and prednisone was started at 50 mg p. o.  leading to a rapid clinical and radiological improvement. IgG4 levels dropped to 0,778 g/L. Following this, prednisone was slowly tapered with recurrence of previously reported abdominal pain and jaundice, as well as radiological progression of the pancreatic masses. A pancreatic biopsy and an elevated IgG4:IgG ratio (>40), together with a IgG4 serum level of 1.92 g/L, confirmed the recurrence of IgG4 related disease (IgG4-RD).Replacement of glucocorticoids with rituximab or methotrexate was discussed, but Methotrexate was selected for health insurance purposes at 25 mg weekly subcutaneously. Glucocorticoid treatment was slowly tapered to 5 mg. The patient remains relapse free to this day, more than 2 years after initiation of methotrexate treatment.3,Some studies suggest an increased risk of malignancies particularly in the first year after IgG4-RD diagnosis.Our case describes rapid increase in PSA after a satisfactory nadir of 0.23 in a favorable intermediate risk cancers. Such increases are very unusual. We speculate that this unusual increase in PSA was due the impaired immune-system caused by his IgG4 disease. We can't prove any such causality and no molecular tests were done to prove such a relationship.IgG4-RD tends to respond well to glucocorticoids in its active inflammatory stage, Second-line treatments include immune-suppressants such as rituximab, azathioprine and methotrexate.+ T cell memory as a result of rituximab-induced B cell depletion, highlighting the essential role for CD4+ cytotoxic lymphocytes in the pathogenesis of IgG4-RD. The role of rituximab in prostate cancer treatment has not yet been established as most antibody immunotherapies have not succeeded in prostate cancer. A 2020 clinical trial, the first such study in eight high-risk prostate cancer patients, neoadjuvant rituximab decreased B-cell and T-cell density in biopsy samples compared to controls, providing evidence that rituximab has the ability to modify the tumor microenvironment in prostate cancer.Rituximab could be an interesting agent to treat both IgG4-RD and his prostate cancer. It is commonly used in the treatment of hematological neoplasms such as CD20-positive leukemia and lymphoma. Rituximab is an anti-CD20 monoclonal antibody and selective B-cell depleting drug. A potential mechanism for such a response involves interference in the maintenance of CD4In conclusion, the presented case shows a fulminant evolution of a prostate cancer with a relatively low aggressiveness treated curatively initially. The rapid rise in PSA and rapid development of metastasis could be due an impairment of the immune system. It can't be excluded that there was much more aggressive cancer in the prostate that was missed on initial biopsy because the patient didn't have a diagnostic MRI before treatment.None.


There is a relative wealth of experience in the initial treatment of IgG4-related disease (IgG4-RD), but little is known about therapeutic measures for recurrent cases combined with multiple organ and tissue involvement.A 43-year-old man with a previous diagnosis of IgG4-RD due to recurrent right lacrimal gland enlargement with eyelid erythema presented with diabetes insipidusWe performed a pituitary Magnetic Resonance Imaging which revealed posterior pituitary rim changes with inhomogeneous enhancement and nodular-like thickening of the pituitary stalk, and performed a water-deprivation-vasopressin test confirmed central diabetes insipidus, and in combination with the patient’s elevated IgG4 levels and past medical conditions, we diagnosed central diabetes insipidus, IgG4-related hypophysitis, and IgG4-RD.After the patient was admitted to the hospital we gave methylprednisolone 500 mg intravenously once daily for 4 days and again for 4 consecutive days after a 10-day interval. During this period combined with mycophenolate mofetil 250 mg twice daily and desmopressin acetate 0.1 mg 3 times daily.The patient was followed up for a sustained period of 6 months and no side effects of glucocorticoid therapy were noted, there were no signs of recurrence, and the daily urine output stabilized in the normal range.We recognized that IgG4 levels do not reflect relapse or long-term control, and that glucocorticoid shock therapy is an optional and reliable treatment strategy for relapsed patients. IgG4-related hypophysitis (IgG4-RH) is a manifestation of IgG4-related disease (IgG4-RD) involving the pituitary gland and is a relatively rare form of pituitary inflammation of varying severity, often with involvement of other organs. We report the clinical data of a patient with IgG4-related pituitary inflammation admitted to our hospital in November 2022, who was seen for diabetes insipidus and had a relapse of IgG4-RD, and whose symptoms improved after glucocorticoid impact therapy. There is a wealth of research on initial treatment, but the choice of medication for patients with relapses is still poorly understood. Based on this case, we reviewed and summarized the relevant literature to improve clinicians’ understanding of this disease.The patient, a 43-year-old male, had recurrent right lacrimal gland enlargement with eyelid erythema in early 2019 with no obvious cause, and his symptoms did not improve significantly after several visits to our ophthalmology department for treatment. Axial CT scan of the orbit suggested slight swelling of the right upper eyelid, bone changes in the right infraorbital wall and local soft tissue density. Further refinement of the immunoglobulin IgG4 assay 1790 mg/dL , which was identified as CDI.am) adrenocorticotropic hormone 21.50 pg/mL (7.20–63.3pg/mL), and (8 am) cortisol 185.00 nmol/L (166–507 nmol/L).We also examined the other hormonal axes associated with the pituitary gland. For thyroid-related, anti-thyroglobulin antibody 11.80 IU/mL (reference range 0–15.00 IU/mL), thyroid-stimulating hormone 4.19 uIU/mL (0.27–4.20 uIU/mL), free triiodothyronine 5.07 pmol/L (3.10–6.80 pmol/L), free thyroxine 14.50 pmol/L (12.00–22.00 pmol/L), and anti-thyroid peroxidase antibody <9.00 IU/mL (reference range 0–34.00 IU/mL). Sex hormone related, prolactin 793.60 mIU/L↑ (86.00–324.00 mIU/mL), estradiol 122.20 pmol/L (99.40–192.00 pmol/L), luteinizing hormone 7.85 mIU/mL (1.70–8.60 mIU/mL), follicle stimulating hormone 12.30 mIU/mL (1.50–12.40 mIU/mL), progesterone 0.38 nmol/L (0.16–0.47 nmol/L), and testosterone 17.35nmol/L (9.90–27.80 nmol/L). Growth hormone 0.28 ng/mL (0.03–2.47 ng/mL), insulin-like growth factor 73.60 ng/mL (60–350 ng/mL),  CDI; (2) IgG4-RD, IgG4-RH; and (3) hypertension grade 1 (low risk). Methylprednisolone 500 mg IV once daily was given on November 4 to 8 and November 17 to 20, 2022, respectively. Combined with MMF 250 mg twice daily and desmopressin acetate 0.1 mg 3 times daily. The patient’s elevated prolactin was considered a pituitary stalk effect and bromocriptine 1.25 mg was given once a night. During the first phase of impact therapy, the patient’s thirst symptoms improved, and urine output gradually changed to 1500 to 2000 mL/day.A repeat pituitary MR enhancement scan on November 21, 2022, suggested that the pituitary stalk nodes were significantly smaller than before Figs.  and 5. A so the search period was from January 1, 2021 to December 1, 2022, and 15 case reports were detected,–16 including 12 with complete clinical information and a total of 24 patients–7,9–14 /European League Against Rheumatism (EULAR) international classification criteria, reviewed 115 cases of IgG4-RH up to 2021 is the largest number of cases included so far, found that IgG4-RH was predominantly male  and predominantly middle-aged and elderly . Diabetes insipidus was one of the common symptoms, and in the 115 cases with posterior pituitary function described in 89 cases, 70 patients had diabetes insipidus (78.7%). Anterior pituitary function was described in 102 cases, of which 86 patients had varying degrees of hypopituitarism and various hormone deficiencies (84.3%). In addition, 87 of them reported specific serum IgG4 levels with a mean of 666.2 ± 936.4 mg/dL.Among them, Amirbaigloo et al,21 while others include primary or secondary tumors, infections, cranial surgical trauma, trauma, autoimmune and, less commonly, genetic factors.,25 In this patient, CDI and lacrimal gland enlargement were the main clinical features, and the serum IgG4 level was significantly elevated, combined with imaging features and good response to glucocorticoid therapy, so the final diagnosis of IgG4-RH was made. It suggests that in clinical practice, the possibility of systemic diseases including IgG4-RD should be considered in CDI with multiple site or organ involvement.CDI refers to varying degrees of polyuria due to reduced release of antidiuretic hormone from various causes, caused by hypotensive or disrupted neuronal function in the supraoptic and paraventricular nuclei of the hypothalamus. The most common cause is idiopathic diabetes insipidus (30%–50%),,27 the exact incidence and prevalence of IgG4-RD is unknown, and current statistics suggest that the disease is slightly more prevalent in middle-aged and older men, and a prospective study conducted in China found a similar gender composition. Involved organs also vary by gender, for example, renal and retroperitoneal lesions are more commonly seen in males, while salpingitis, lacrimation and primary biliary cirrhosis are more prevalent in females. For the patient in this case, we also conducted some further examinations and found no involvement of other organs.IgG4-RD is a class of fibroinflammatory diseases in which involvement of almost every organ has been reported. but the exact etiology is unclear. Mattoo et al found clonally proliferating CD4+ cytotoxic T cells in both the peripheral blood and the lesions of the patients, and they hypothesized that these cells were key to the pathogenesis. (Fig.  suggesting that IgG4 antibodies themselves are not pathogenic and that their overexpression may be the response to primary inflammatory stimuli.For the pathogenesis of IgG4-RH, or more generally IgG4-RD, it is currently, believed that there is a role for both genetic risk factors, bacterial infection, and autoimmunity,is. Fig.  The pati mentioned above. It should also be emphasized that pathological changes are an important basis for the diagnosis of IgG4-RD. The typical pathology is a dense lymphocytic tissue infiltration with storiform pattern, which may be accompanied by obliterative phlebitis and mild to moderate eosinophilic infiltration, and this pathological change is similar between the different organs involved.The fact that no biopsy was performed during the treatment of this patient is a regret of the whole treatment process, but combined with the patient’s previous history of IgG4-related ophthalmopathy and the available tests, we can exclude CDI caused by primary or secondary tumors, infections, cranial surgical trauma, etc., and the diagnosis of IgG4-RH is clear in accordance with the diagnostic criteria for IgG4-RH proposed by Leporati et al and most of the original case reports were for cases with initial onset. We emphasize that recurrence of IgG4-RD may involve more organs and tissues, and in this case, the patient had a new onset of pituitary inflammation and CDI based on long-term control of the primary symptoms. There are not many references for the treatment of relapsed patients. Of the 24 patients we summarized, 3 patients who reported recurrence during follow-up, but the description of treatment for the relapses was less clear in all cases. In this case, the patient’s symptoms improved significantly after methylprednisolone impact therapy combined with MMF, suggesting that glucocorticoids are still effective in relapsed patients and that impact therapy is an optional dosing modality.In terms of treatment, IgG4-RH also follows the IgG4-RD approach, with guidelines recommending glucocorticoids as the first-line treatment in the presence of symptomatic activity. The natural course and prognosis of IgG4-RD are not fully understood, but there is a clear tendency for IgG4-RD to recur, In the initial treatment, drug combinations have been shown to provide greater benefit, and a similar therapeutic effect may have been seen in this case of relapse. In addition, considering the tendency of IgG4-RD to recur, patients need to be followed up regularly over a long period of time, and they also need to be able to report new symptoms in a timely manner.In conjunction with the above, we recommend that in patients with relapses and new organ involvement, methylprednisolone impact therapy can be considered at a dose of 500 to 1000 mg per day, with a lower dose chosen when combined with immunosuppressive drugs for 3 days, followed by a 10% to 20% reduction every 1 to 2 weeks, gradually decreasing to a maintenance dose that prevents relapse. In addition, some patients were not treated with glucocorticoids during their previous therapy and can be discontinued directly after the effect of shock therapy within 2 weeks, which is unlikely to trigger suppression of the hypothalamic-pituitary-adrenocortical axis. the disease has not been recognized for a long time. Combined with the literature review above, the epidemiology, pathogenesis and treatment of IgG4-RH need to be further improved, and there are not many references for the treatment of relapsed patients in clinical practice. We recognize from the treatment of this patient that IgG4 levels do not reflect relapse or long-term control, and that glucocorticoid shock therapy is an optional and reliable treatment strategy for relapsed patients, the treatment process of this case is expected to enrich the knowledge of disease treatment.Overall, IgG4-RD is a class of fibrous inflammatory diseases that can involve multiple organs throughout the body, and the involvement of different organs can occur sequentially. Pituitary gland involvement can result in CDI, anterior hypopituitarism or total hypopituitarism, and glucocorticoids are currently the recommended effective treatment. Although the number of reported cases of IgG4-RH has gradually increased since the first case in 2004,Conceptualization: Yongzhuo Yu.Supervision: Yangang Wang.Writing – original draft: Yongzhuo Yu.Writing – review & editing: Lili Xu, Yunyang Wang, Wenxuan Li.


The disposal of glass fiber-reinforced plastic (GFRP) waste has become an urgent issue in both the engineering and environmental fields. In this study, the feasibility of reusing mechanically recycled GFRP in concrete was evaluated. Secondary screening of the recycled material was conducted to obtain different types of products, and the recycled GFRP (rGFRP) was characterized. Subsequently, the effect of rGFRP on concrete performance was evaluated using different dosages  of rGFRP powder and rGFRP cluster (with different sizes and fiber contents) to replace fine aggregate in concrete preparation. The experimental results indicated that the addition of rGFRP powder has no significant impact on the mechanical properties of concrete, while the addition of a small amount of rGFRP cluster slightly improves the compressive strength and splitting tensile strength of concrete. Additionally, the short fibers in rGFRP improve the failure mode of concrete, and increased fiber content and longer fiber length demonstrate a more pronounced reinforcing effect. The challenges and potential directions for future research in the realm of reusing rGFRP in concrete are discussed at the end. A systematic process for reusing GFRP waste in concrete is proposed to address the primary challenges and provide guidance for its practical engineering application. In recent years, the use of glass fiber-reinforced plastic (GFRP) has been widely adopted in the aerospace, construction, and automotive industries ,3,4,5,6.Many countries have imposed restrictions and prohibitions on traditional landfilling and incineration methods . TherefoAt present, mechanical recycling has emerged as the most practical and feasible method for managing GFRP waste due to its low cost, minimal environmental impact, and capability to efficiently process large quantities of GFRP waste . The mecThe construction industry has made significant efforts to reduce waste and promote recycling to mitigate adverse environmental impacts ,21,22,23Asokan et al.  found thThe results from the previously mentioned studies reveal significant differences. Even when the amount of GFRP waste added is similar, the impact on concrete’s mechanical properties varies distinctly. This variation can be attributed to (1) differences in GFRP waste size, including particle size and fiber length, resulting from various recycling processes, and (2) differences in the composition of GFRP waste from different sources, particularly in terms of fiber content. Therefore, it is necessary to systematically evaluate the effect of these factors on concrete performance.In this study, an experimental study was conducted to investigate the effects of mechanically recycled GFRP with different sizes, compositions, and contents on the performance of concrete, and to evaluate its feasibility of reuse in concrete. Subsequently, the challenges faced by reusing GFRP waste in concrete at the current stage were analyzed, and existing reuse methods were summarized and optimized, enabling us to propose a systematic reuse program.The rGFRP used in this study was mechanically recycled from decommissioned wind turbine blades. GFRP material is composed of E-glass fiber and unsaturated polyester resin. After the GFRP waste was crushed and sieved, secondary screening using a 1.18 mm sieve was conducted to obtain two types of recycled materials, namely, rGFRP powder and rGFRP cluster. As shown in Seven different concrete mixes were used in this study, as outlined in The process of specimen preparation and testing is outlined in 2 solution at room temperature until the testing day. For each type of concrete, a total of twelve cubes, measuring 100 mm × 100 mm× 100 mm, were prepared.Following the mixing process, the fresh concrete was tested for slump according to the standards outlined in GB/T 50080 . The conAfter the concrete attained the appropriate curing age, a series of performance tests were conducted, including mechanical property and density tests, on the hardened concrete. A summary of the test specifications and specimen sizes can be found in The composition of rGFRP was analyzed using a simultaneous thermal analyzer . Additionally, the microscopic morphology of rGFRP was characterized using scanning electron microscopy .The mass loss curves for the rGFRP are depicted in The microscopic morphology of rGFRP is shown in Based on the characterization, it is evident that the two recycled materials exhibit varying proportions of glass fibers and organic resins. Additionally, distinctions are observed in particle size (powdered component) and fiber length (fibrous component).The workability of concrete is a crucial performance indicator that depends on the amount of water added during concrete mixing. This study compared the slump of different test groups to examine the influence of rGFRP type and content on concrete workability. All test groups had the same w/c ratio, ensuring uniform water content during concrete mixing.Before casting the test specimens, slump tests were conducted on each mixture. The slump was measured for six different batches of concrete for each mixture to reduce errors caused by manipulation or other factors. The test results are shown in The slump of the concrete decreased linearly with an increase in rGFRP content. This outcome aligns with findings by other researchers, who have found that the high specific surface area of rGFRP, compared to natural sand, necessitates a larger surface to be covered by a water film during mixing. This leads to a decrease in free water in fresh concrete and an increase in the water demand for concrete . From a Additionally, the negative effect of rGFRP clusters on the concrete slump was more pronounced due to their longer fiber length and higher fiber content, leading to higher yield stress and plastic viscosity of fresh concrete.The compressive strength of CC and rGFRP concrete cube specimens was tested after 7 and 28 days of curing using a universal testing machine , following the procedures specified in GB/T 50081 . The speThe compressive failure mode of the concrete is shown in The results of the compressive strength tests are shown in The 28-day compressive strength values of concrete containing rGFRP powder were similar to those of control concrete without rGFRP (43.2 MPa), indicating that rGFRP powder had no significant effect on the 28-day compressive strength of concrete. Conversely, the influence of the rGFRP cluster on the 28-day compressive strength of concrete was more noticeable. With its content of 1%, the 28-day compressive strength of concrete was 46.1 MPa, representing a 6.8% increase. However, when the content was further increased, the compressive strength decreased. Specifically, the compressive strength decreased from 41.7 MPa to 38.5 MPa as the content was increased from 3% to 5%.2O3, SiO2, and other polymers in rGFRP can strengthen the compressive strength of concrete, and the short fibers in rGFRP can inhibit the development of concrete cracks [These phenomena can be attributed to several factors: (1) The presence of CaO, Ale cracks ,41. (2) e cracks , leadingA is the area of the splitting surface of the specimen, and F is the maximum load recorded at the time of concrete failure. The concrete splitting tensile failure mode is shown in Splitting tensile tests were conducted on concrete cube specimens according to GB/T 50081 . A stripThe control group specimen exhibited single crack initiation under tensile load. As the load increased, the crack initially initiated at the center of the cross-section of the specimen, and then, propagated towards the edge strip. However, concrete specimens containing rGFRP powder and rGFRP cluster exhibited multi-crack initiation with similar failure modes. In addition to the main through-crack located at the center of the section, numerous branch cracks also existed. These branch cracks were shorter in length and did not traverse the entire surface of the concrete. The addition of more rGFRP resulted in a more pronounced multi-crack morphology. This indicated that the anisotropic microstructure of material properties, caused by the short fibers in rGFRP, changed the stress distribution and crack expansion direction. This dispersed crack expansion plays a restraining role in the main crack expansion.As shown in During the splitting tensile test of the rGFRP concrete specimens, the load–displacement curves exhibited two types of curve, as shown in Furthermore, it was observed that the load–displacement curves exhibited by the concrete specimens of group C05 displayed distinctive behavior characterized by a second type of curve. The ascending portion of this type of curve shared similarities with that of the first type of curve. However, following the attainment of its peak, the load curve demonstrated a secondary peak after a period of decline. This phenomenon is often referred to as strain hardening. Subsequently, the load rapidly decreased until the concrete completely failed due to damage.The underlying mechanism is as follows: When the applied load peaks and the concrete develops cracks, the stress is transferred to the fibers. Consequently, more fibers participate in the load-bearing process following cracking. The bond between these fibers and the matrix material creates an anchoring effect, thereby increasing the concrete’s load-bearing capacity. Simultaneously, some of the fibers break under the influence of the load, absorbing a certain amount of energy. However, when the fibers are pulled out or broken, they lose their load-bearing capacity, and the curve experiences a rapid descent.Due to the limited length and number of fibers, the descending portion of the curve after reaching the peak is not gradual. This indicates that the reinforcing effect of these short fibers on the matrix is not optimal.To examine the mechanism of short fibers in rGFRP on the concrete microstructure, the small fragments that broke off during the compression and splitting tensile tests on rGFRP concrete were analyzed. The observation results are shown in Upon examining the SEM images, it was observed that more fibers were pulled out than broken, suggesting that the bond between the fibers and the matrix was not robust, as shown in 3. As the amount of rGFRP increased, the density of the concrete decreased linearly. Since the density of rGFRP is lower than that of fine aggregate, increasing the content of rGFRP in concrete will lead to a reduction in the density of concrete.To investigate the effect of rGFRP on the density of concrete, the dry density of the concrete was calculated after 28 days of curing. To minimize errors, 12 concrete specimens were selected for density measurement in each group. The results, shown in When the rGFRP content was 3%, the compressive strength and splitting tensile strength of concrete improved, and the density of concrete reduced by 0.66% and 1.74%, respectively. Replacing 3% of fine aggregate with rGFRP in concrete appears to be a favorable option since it slightly increases strength while reducing density. When the rGFRP cluster content was 5%, the strength of the concrete decreased slightly, but the density of the concrete reduced by 3.93%, significantly decreasing the weight of the concrete structure. Therefore, in practical applications, determining the optimal dosage of rGFRP should be based on its intended use.While some progress has been made in researching the mechanical recycling of rGFRP, its reuse in concrete has been primarily limited to laboratory research. Different scholars have focused on various aspects of this research, thus resulting in conclusions that may not be universally applicable. As a result, there appears to be a gap between the current state of research and its practical implementation in the industry. Yazdanbakhsh and Bank  believedThere is a lack of a widely applicable program for reusing GFRP. Building upon this study and other pertinent research, we propose an optimized rGFRP reuse program to address this gap. As shown in Currently, there are three ways to reuse rGFRP obtained through mechanical recycling in concrete. One approach involves processing GFRP waste into granules, which can serve as a fine aggregate replacement in concrete. Another method involves cutting GFRP waste into large pieces, suitable for use as a coarse aggregate replacement in concrete. Furthermore, GFRP waste can also be employed as fiber-shaped reinforcing bars by modifying the mechanical recycling processes. All three options for using rGFRP in concrete are attractive. Replacing aggregate comprising the largest proportion of concrete with rGFRP can maximize the reuse of GFRP recycled materials. Fiber-shaped rGFRP, retaining its relatively intact structure and performance, can be regarded as weakened fibers and has a significant enhancing effect on the mechanical properties of concrete.Yazdanbakhsh et al.  conducteFu et al.  developeThe results of prior studies in this research indicate that the impact of rGFRP on concrete properties varied according to the particle size levels. Likewise, Mastali et al.  observedRecycling plants that process waste from various sources of GFRP produce waste materials with different compositions. When incorporated into concrete, these waste materials can significantly affect the properties of the concrete, as shown in a study by García et al. , where sThe recycled waste should be tested to (a) measure its physical properties, (b) determine the type and content of its components, and (c) characterize its microstructure. Testing methods may include sieve analysis or laser particle size determination to determine particle size distribution, thermal analysis, and spectroscopic techniques such as XRD, XRF, and FT-IR to determine the type and content of fibers, resins, and other components in rGFRP, and scanning electron microscopy to determine the distribution of fibers and other components. Based on the data obtained from the tests and the properties of concrete containing GFRP waste, the usable range of the material can be determined.Currently, there are limited studies in this area, and the performance of the raw materials used varies greatly. There is a lack of qualitative and quantitative understanding of the effects of the chemical composition and microstructure parameters of rGFRP on concrete performance.Mix proportion optimization aims to improve the performance of concrete and maximize the consumption of rGFRP while specifying performance requirements. Yazdanbakhsh and Bank  acknowleVarying mix proportions should be used for rGFRP concrete with different rGFRP content to ensure the desired workability and mechanical properties of the concrete, as well as to determine the optimal dosage of different types of rGFRP and their corresponding mix ratio. Optimization methods may include the use of superplasticizer, the addition of fly ash, adjustment of the w/c ratio, and the optimization of aggregate grading, among others.With the development of computer technology, machine learning has been widely applied in the field of concrete mix design ,46,47,48The performance of rGFRP concrete exhibits significant variation due to the inherent variability in concrete's internal structure and the uneven distribution of rGFRP within it. So, it is essential to establish the quantitative relationship between factors such as the size and dosage of GFRP waste and the mechanical properties of concrete, which will determine their application in practical engineering projects. Currently, there is limited research in this direction, yet it is crucial because it can provide guidance for the mechanical recycling process of GFRP and the overall reuse of GFRP waste in concrete.Apart from evaluating the workability and mechanical properties of concrete, it is crucial to emphasize its durability, especially in alkali–aggregate reaction testing. The alkali in cement can react with the silica in GFRP waste, leading to an alkali–silica reaction (ASR) that triggers destructive expansion and shortens the lifespan of concrete. Hence, thorough testing and evaluation of the alkali–aggregate reaction is necessary to ensure the durability and long-term performance of the rGFRP concrete. Moreover, feasible methods and technologies, such as adding mineral admixtures to suppress the alkali–aggregate reaction, need to be explored and applied to maximize the reliability and durability of rGFRP concrete.GFRP products are increasingly being used in various industrial fields. However, the speed of GFRP waste accumulation is continuously increasing, which necessitates the development of more sustainable solutions within the GFRP industry. In light of this, this study conducted experiments to assess the reuse potential of recycled GFRP in concrete. The following conclusions were drawn from the experiments:The addition of rGFRP reduced the slump of concrete. However, at lower content levels, the effect was negligible. The influence of a low content (1–5%) of rGFRP powder on the strength of concrete was minimal. The addition of a small amount of rGFRP cluster slightly improved the compressive strength and splitting tensile strength of concrete. The presence of short fibers in rGFRP improved the compressive and splitting tensile failure mode of concrete. Moreover, higher fiber content and longer fiber length exhibited a stronger reinforcing effect.The addition of rGFRP decreased the density of concrete, indicating that it can somewhat facilitate the lightweight design of concrete structures.To guarantee the quality and performance of concrete when using various GFRP waste materials, this study discussed and optimized existing reuse methods. It proposes an optimized procedure for GFRP reuse to maximize the technical and economic benefits. This procedure aims to facilitate the reuse of GFRP waste in engineering applications and advance its industrial-scale implementation.Currently, there is a lack of understanding regarding the microstructural mechanisms underlying the impact of rGFRP on the mechanical properties of concrete. Further research is required to delve into this aspect, which will help advance the practical application of GFRP waste in engineering projects.


This study was conducted to establish a rat model of acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) using the combination of bleomycin (BLM) and lipopolysaccharides (LPS).Twenty-four male Sprague Dawley rats were allocated into two equal groups: the sham or the bleomycin and lipopolysaccharides-induced AE-IPF group (BLM-LPS). On Day 7, BLM intratracheally and LPS intraperitoneally were both used to administer AE-IPF. The BLM-LPS group and its respective sham group were terminated on Days 8, 14, or 21. Samples of bronchoalveolar lavage fluid (BALF) and lungs were taken and investigated for cell count and histopathology.On Day 8, histological analysis revealed inflammatory cell infiltration with edema and hyaline membrane, and the BALF differential cell count revealed high neutrophil counts. By having a higher collagen density area and Ashcroft modified score than the sham group on Day 14, the BLM-LPS group displayed significantly lower oxygen saturation, alveolar air area, and a fibrotic appearance. However, there was a spontaneous resolution in inflammation and fibrotic appearance on Day 21 after the BLM administration.By combining BLM and LPS, it was possible to create a successful rat model of AE-IPF. The present model showed the peak exacerbation on Day 8 and the fibrotic peak on Day 14, which gradually improved. The optimal time for the new AE-IPF therapeutic intervention was determined to be between Days 8 and 14. Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease of unknown etiology of the respiratory system that is limited to lung tissue . PatientA suitable animal model is needed to identify a potential new therapy for AE-IPF. An animal model is expected to be able to mimic a condition or disease in humans and its manifestations. Although animal models cannot reproduce all of the variables seen in human illness, they may give insight into the pathobiology of tissue damage, cellular inflammation, immunological control, and the recovery process ,12. The Among the many agents to induce IPF, bleomycin-induced lung fibrosis is the most widely used in animal models. Bleomycin (BLM) is an anticancer agent with significant pulmonary toxicity; thus, BLM has been commonly used in preclinical testing ,14. BleoAfter obtaining ethical clearance from the Ethics Committee of the Faculty of Medicine, Universitas Indonesia (KET-517UN2.F1/ETIK/PPM.00.02/2021), this experiment was performed at the Indonesian Medical Education and Research Institute.®) was obtained from PT Kalbe Farma, Tbk (Indonesia). LPS from Escherichia coli O55:B5 were purchased from Sigma Aldrich, St. Louis, Missouri (cat no. L2880). Ketamine HCl (KTM-100®) was purchased from Guardian Pharmatama (Indonesia). Xylazine for injection (Xyla®) was purchased from Interchemie (Holland). NaCl (0.9% solution) was purchased from B Braun Pharmaceutical (Indonesia). Natural Buffered Formalin 10% was purchased from BBC Chemical (USA). All other chemicals and solvents were analytical grade.Bleomycin hydrochloride for injection  were obtained from the National Agency for Drug and Food Control’s Animal Breeding Facility at the age of 8–10 weeks. The rats used in the study were acclimatized for 2 weeks before the experiment started. The rats were placed in a room at a temperature of 25°C–27°C and a humidity of 45%–65%, 12-h light and dark cycles, standard pellet food, and unlimited water.Twenty-four rats were randomly divided into two groups of 12: the sham group and the bleomycin-lipopolysaccharides (BLM-LPS) group. Rats were anesthetized intramuscularly with ketamine (75 mg/kg BW) and xylazine (5 mg/kg BW) before receiving intratracheal administration of saline (sham group) or BLM (BLM-LPS group). The rats in the sham group were incised in the trachea region and stitched back, followed by the administration of 0.9% saline solution intraperitoneally on Day 7. In the BLM-LPS group, 5 mg/kg BW BLM was injected intratracheally with a 30-gauge needle, followed by air. The rats were shaken several times to ensure that BLM was distributed evenly throughout both lung tissues. On Day 7, after BLM, the rats in the BLM-LPS group received an LPS injection (7.5 mg/kg BW) intraperitoneally. Both groups of rats were randomly terminated three times, on Days 8, 14, and 21, to observe differences in disease progression over time following BLM-LPS induction.On the day of animal sacrifice , rats were anesthetized using ketamine (75 mg/kg BW) and xylazine (5 mg/kg BW) via the intramuscular route. After being anesthetized, oxygen saturation was checked in the rats using an animal pulse oximeter. A probe to measure oxygen saturation was placed on the rats’ leg. Blood samples were collected via heart puncture. The neck and thoracic cavities were opened, a 22-gauge intravenous catheter was injected into the trachea, and the bronchoalveolar lavage fluids were collected. The needle was pulled out, and the plastic tube was left to connect to a syringe containing 2–3 ml of saline. Saline was slowly injected into both lungs. While saline was injected, the rat’s body was slightly lifted, and the rat’s nostrils were closed with a finger to ensure no fluid came out of the nose. This procedure was repeated three times, and the BALF was collected in the same tube. Afterward, the lungs were harvested, rinsed in saline, dried on paper towels, and weighed to calculate the lung/body weight (BW) index. Lungs were immersed in 10% neutral-buffered formalin for histopathological examination.g for 20 min was used to separate the BALF from each rat. Phosphate-buffered saline was used to resuspend sedimentary cell pellets. The procedure was divided into two parts: the cell count procedure and the cytospin procedure. The improved Neubauer counting chamber was used to determine cell counts, and trypan blue exclusion was used to determine viability. For 5 min, the cytospin procedure was performed at 1,500 rpm. Giemsa staining was performed after the cytospin procedure (Cytospin 4 Cytocentrifuge Epredia). The differential cell count was assessed using a light microscope (Leica DM 1000 LED) on Giemsa-stained slides.Cold centrifugation at 1,000xThe lung tissues were embedded in paraffin wax, sectioned (5 μm), and stained with HE and Masson’s trichrome after being placed in a vial containing 10% neutral-buffered formalin. Tissues were examined using a Leica DM 1000 LED microscope. HE stains were used to examine cell morphology and inflammation. Collagen fibers in tissues were detected using Masson’s trichrome staining. Hubner et al.  adapted 2), corresponding to the total lung region (μm2) in the ROI. The ROI area did not include bronchi and blood vessels [ImageJ 1.53t analysis software was used to determine the degree of fibrosis. Regions of interest (ROIs) were chosen randomly using a 100× magnification and three images per slide. An image analysis application was used to identify regions of green collagen in each ROI using similar contrast, intensity, and color threshold settings . The col vessels .2) occupied by air was expressed as a percentage of the total lung area (μm2) in ROI. The ROI area did not include bronchi and blood vessels [Alveolar air area was measured as an indirect fibrosis parameter. A white threshold was used to detect the alveolar air area within the ROIs using ImageJ 1.53t analysis software, and the same ROIs were used for counting collagen content. The percentage area . A p-value of less than 0.05 was considered significant.The mean and standard error of the mean (SEM) were used for presenting the data. Multiple Following bleomycin administration, rats given BLM-LPS had a slight decrease in BW compared to rats in the sham group . On Day The lung index was determined by dividing the lung weight by the total BW of the rats. Higher lung indexes were seen in the BLM-LPS-induced animals compared to the sham group. On Day 8, the rats had the most significant lung index, followed by Days 14 and 21 .Analysis of BALF with neutrophil marked increase in the BLM-LPS groupCell counts in BALF were examined on the same day as the rat necropsy. The number of cells increased significantly in the BLM-LPS group terminated on Day 8, then decreased slightly in the BLM-LPS group terminated on Day 14, and returned to normal on Day 21 .Most cells in the differential count in the sham group were macrophages, according to BALF examination using cytospin followed by Giemsa staining . HoweverMarked inflammatory cell infiltration on Day 8 in the rats administered with BLM-LPS Large lung fibrotic area on Day 14 in the rats administered with BLM-LPS Improvement of inflammation and fibrosis on Day 21 in the rats administered with BLM-LPS Persistent increase in Ashcroft modified scores and collagen density area as well as a decrease in alveolar air area in the BLM-LPS group on Days 8, 14, and 21The Ashcroft modified score increased significantly in all bleomycin-lipopolysaccharide groups on Days 8, 14, and 21 . The higThe ImageJ application measured the green-colored areas on lung slides stained with Masson’s trichrome to determine collagen density. The area of collagen density increased with BLM and LPS induction, reaching a peak in rats on Day 14. On Day 21, there was a decrease in collagen density, indicating that the rat lung injury had started to resolve .The alveolar air area can be seen on lung slides stained with Masson’s trichrome in the ImageJ application by measuring the white area. In BLM and LPS-induced rats, alveolar air area decreased for 8, 14, and 21 days, with a maximum decrease at 14 days .Patients with IPF may experience life-threatening acute exacerbations called AE-IPF. AE-IPF has a poor prognosis and a high morbidity and mortality rate, which can lead to increased hospitalization rates. Reduced lung function, impaired oxygenation, and a high fibrosis score increase the risk of death in patients with AE-IPF .The molecular pathogenesis of IPF is not fully understood. However, epithelial injury and poor wound healing are thought to play essential roles in this disease, in addition to chronic inflammation. Old age, exposure to cigarettes and asbestos, comorbid diseases, such as diabetes mellitus, gastroesophageal reflux, and obstructive sleep apnea, and infections are all risk factors for IPF .AE-IPF pathobiology is a combination of chronic elements such as epithelial cell malfunction, fibroblast buildup and activation, and other significant factors such as acute stress and acute lung damage. Critical events such as infection or microaspiration induce acute broad lung damage, characterized by the production of hyaline membranes and interstitial edema in the early stages .The lack of animal models that mimic AE-IPF cases creates barriers to advancing AE-IPF research. Rats have been used in drug research for a long time because they have larger bodies and more blood volume than mice. A suitable rat model enables a more thorough examination of test parameters. As a result, we intend to create an AE-IPF rat animal model for future research.The use of intratracheal BLM to create pulmonary fibrosis animal models has been established and widely used in various studies ,26. BLM In this study, the administration of LPS on Day 7 was intended to mimic AE-IPF in humans by acting as an infection that causes an acute exacerbation state. Our study showed that using BLM followed by LPS resulted in a good model of AE-IPF with an optimal window between Days 8 and 14 after BLM administration. The administration of BLM alone had little effect on the BW of the rats. However, drastic weight loss was observed in the rats after administration of LPS, which was intended to mimic acute exacerbations; however, the effect was transient and tended to resolve at Days 14 and 21. LPS  is the primary trigger of lung inflammation in experimental animals. It is commonly accepted as a reliable inducer of acute exacerbation in lung diseases ,29.The research findings were consistent with those of Miyamoto et al.  who repo2 and pCO2 had returned to normal, indicating that breathing had improved. Macroscopic and microscopic examinations of BLM-LPS rats terminated on Day 14 revealed more extensive lung structural damage, with inflammatory cell infiltration and fibrotic tissue overlying the alveolus. However, when BLM-LPS rats were terminated on Day 21, the inflammatory cell infiltration in the lungs decreased, as did the fibrotic tissue. This image shows that there has been an improvement on Day 21, while the fibrotic peak occurs on Day 14.The oxygen saturation was the lowest in BLM-LPS-induced rats on Day 14, yet it improved on Day 21. The improvement in oxygen saturations indicates that spontaneous resolution has occurred as time has elapsed. This is consistent with the findings of Miyamoto et al. , who disDespite some discrepancies in the results, numerous studies have shown that the acute IPF worsening brought on by BLM-LPS is partially reversible , 32, 33.This study found an increase in lung index in BLM-LPS rats terminated on Days 8, 14, and 21, which was due to the increment in the weight of the rat lungs. This suggests that inflammatory cell infiltration and fibrosis in the lung increase lung weight. In an animal model of acute lung injury, Song et al.  reportedThe findings confirmed that when LPS is administered on Day 7, it causes an acute exacerbation condition with a peak effect on Day 8. The BALF cell count dramatically increases on Day 14 and gradually decreases on Day 21. On Day 8, the differential BALF count with Giemsa stain revealed that neutrophils were elevated following LPS administration. This indicates a severe infection. This is similar to the AE-IPF condition in humans, where neutrophils are reported to be the predominant cell type in BALF. According to Schupp et al. , there wIn HE staining of BLM-LPS rats on the eighth day, there was an infiltration of inflammatory cells covering the alveoli, pulmonary edema, and the formation of hyaline membranes, which corresponded to the description of AE-IPF cases in humans . AdminisThe limitation of this study includes the relatively short period of time over which we monitored the resolution of pulmonary fibrosis. We suggest that the study be extended to 28 days after BLM injection. Furthermore, further tests may be performed in animal models to measure the amount of inflammation and fibrosis. Though BLM-LPS-induced rats are a good model for understanding the progressivity of the disease, they could not fully replicate the irreversible course of fibrosis seen in patients . TherefoBased on all the above findings, acute exacerbations occurred on Day 8, 1 day after LPS administration, with fibrosis peaking on Day 14. These findings were supported by an increase in collagen density area and a decrease in alveolar air area found in BLM-LPS rats terminated on Day 14. The disadvantage of using BLM to induce IPF is the possibility of spontaneous resolution . AccordiThe rat animal model of AE-IPF was successfully obtained by administering a combination of BLM and LPS. In the present rat AE-IPF model, a new intervention candidate can be administered between Days 8 and 14 when there is an acute exacerbation and fibrotic peak, and no spontaneous resolution has occurred between the periods.


Background: Irrational antibiotic use among hospitalized patients can lead to antibiotic resistance. For rational use, the WHO introduced the Access, Watch, and Reserve (AWaRe) classification of antibiotics. We explored antibiotic use according to the AWaRe classification among patients hospitalized with severe acute respiratory infection (SARI) between the prepandemic and COVID-19 pandemic periods in Bangladesh. Methods: From June 2017 to November 2022, we analyzed SARI inpatient data from the hospital-based influenza surveillance platform at 9 tertiary-level hospitals in Bangladesh. We defined June 2017–February 2020 as the prepandemic period and March 2020–November 2022 as the pandemic period. Physicians identified inpatients meeting the WHO SARI case definition and recorded patient demographics, clinical characteristics, and antibiotics received during hospitalization. We used descriptive statistics to summarize the data. Results: We enrolled 20,640 SARI patients ; and among them, 18,197 (88%) received antibiotics (26% of those received >1 different course of antibiotics). Compared to the prepandemic period, the proportion of antibiotic use among SARI patients was higher during the pandemic: 93%  versus 83%  (P < .001). According to AWaRe classification, Access, Watch, and Reserve groups accounted for 32% , 86% , and 0.05% (n = 4), respectively, before the pandemic and 32% , 90% , and 0.08% (n = 8), respectively, during the pandemic . The most common antibiotic prescribed for children aged <5 years during the prepandemic was ceftriaxone , followed by amikacin  and flucloxacillin ; similarly, during the pandemic, most common antibiotic prescribed was ceftriaxone , followed by amikacin  and flucloxacillin . The most common antibiotic prescribed for patients aged ≥5 years during the prepandemic period was ceftriaxone , followed by amoxicillin-clavulanic acid  and azithromycin . During the pandemic, the most common antibiotic prescribed for patients aged ≥5 years was ceftriaxone , followed by amoxicillin-clavulanic acid  and clarithromycin . Among children aged <5 years, use of the Watch group of antibiotics during the prepandemic and pandemic periods was similar: 94%  versus 95%  (P = .099). However, among patients aged ≥5 years, the use of Watch antibiotics was higher during the pandemic compared to the prepandemic period: 87%  versus 82%  (P < .001). Conclusions: Use of antibiotics in the Watch group was predominant among SARI patients both before and during the COVID-19 pandemic, and it increased among SARI patients aged ≥5 years during the pandemic period in Bangladesh. Promoting antibiotic stewardship programs for physicians, including in-service training on antibiotic use, could reduce irrational antibiotic use, which might contribute to mitigating antibiotic resistance in the country.Financial support: This study was funded by the CDC.Disclosures: None