In the first series of kidney biopsies, several gene families known for metabolizing reactive aldehydes, such as aldehyde dehydrogenase (ALDH), aldo-keto reductase (AKR), and glutathione-S transferase (GSTA), were upregulated in kidneys that did not develop delayed graft function versus those that did. In the second series of kidney grafts, we focused on measuring aldehyde-induced protein adducts and ALDH enzymatic activity. The reactive aldehyde metabolism by ALDH enzymes was reduced in kidneys with delayed graft function compared to those that did not . ALDH enzymatic activity was also negatively correlated with length of hospital stay after a kidney transplant. Together, our study identifies a reduced ALDH enzymatic activity with kidneys developing delayed graft function compared to those that did not. Measuring ALDH enzymatic activity and reactive aldehyde-induced protein adducts can potentially be further developed as a biomarker to assess for delayed graft function and recovery from a kidney transplant.Delayed graft function is an early complication following kidney transplantation with an unclear molecular mechanism. Here we determined whether impaired reactive aldehyde metabolism is associated with delayed graft function. Human kidney biopsies from grafts with delayed graft function were compared with grafts that did not develop delayed graft function by Ingenuity gene pathway analysis. A second series of grafts with delayed graft function ( Organ transplantation, including kidney transplantation, is a known and planned occurrence of organ reperfusion injury. Reperfusion injury in turn can trigger delayed graft function, resulting in the deferred functional recovery of the donor kidney following kidney transplantation.Delayed graft function detrimentally affects renal function and graft longevity and is a risk factor for acute kidney rejection , 2. The During reperfusion of an organ, reactive oxygen species produced at the mitochondria causes lipid peroxidation which generates reactive aldehydes which can impair cellular functions –9. The rAlthough kidney transplants from living donors are less susceptible to delayed graft function when compared to grafts from deceased donors , the difThe study protocol was approved by the medical ethics committee at the Leiden University Medical Center. Written informed consent was obtained from each patient.Paired renal cortical biopsies were obtained at the end of the cold ischemic period (prior to implantation) and 45 min after reperfusion of the kidney in the recipient. Kidney biopsies from an initial 18 donor kidneys were used to conduct the whole genome array portion of the study . We thenRenal allografts were perfused and stored with either University of Wisconsin solution or Custodiol® HTK (histidine–tryptophan–ketoglutarate) solution. None of the grafts were machine perfused. For renal transplantation, all patients were induced by propofol, sufentanil, and atracurium. Patients were intubated in addition to a central venous catheter used for intraoperative monitoring. Patients received basiliximab (day 0 and 4) as immunosuppressive induction. Patients were maintained on tacrolimus or cyclosporine A, mycophenolate mofetil, and steroids for immunosuppression.Biopsies were taken from the upper pole of the kidney. For biopsies taken after reperfusion, a spring-loaded automatic biopsy needle was used (16 Ga Travenol). Tissue was snap frozen in liquid nitrogen and stored at −80°C and labeled with a unique identifier, blinding the person performing the analysis. For the genome array studies, all samples were analyzed together after the groups were collected. The validation studies were also performed after the tissue was obtained, selectively using only tissue from deceased donors who developed delayed graft function after transplantation.The transplanted patients were also followed during their hospital stay. The length of hospital stay after transplantation in addition to glomerular filtration rate at postoperative day 7 was also assessed. Patients were considered as developing delayed graft function if they were in need of dialysis within the first week after transplantation. For those requiring dialysis, acute rejection was excluded as a cause of delayed graft function by renal biopsy. Surgical complications of the transplant procedure were also excluded as a source of delayed graft function. Further, if transplant recipients required one episode of dialysis after transplantation due to incident hyperkalemia, these grafts were also not included as having delayed graft function.Paired biopsies were taken for the patients undergoing renal transplantation with one biopsy prior to transplantation and one biopsy 45 minutes after transplant reperfusion. From these renal biopsies, total RNA was extracted using RNAzol  and glass beads. The integrity of each RNA sample was examined by Agilent Lab-on-a-chip technology using the RNA 6000 Nano LabChip kit and a Bioanalyzer 2100 . RNA preparations were considered suitable for array hybridization only if samples showed intact 18S and 28S rRNA bands and displayed no chromosomal peaks or RNA degradation products (RNA integrity number >8.0). Microarray analysis was performed using Illumina whole-genome gene expression BeadChips  according to the manufacturer's instructions at the Service XS facility in Leiden.The tissue biopsies collected from the two groups were run on separate gene arrays. The 2 paired tissue biopsies obtained for each transplanted kidney resulted in running 36 gene arrays for the tissue biopsies that were collected. The gene arrays obtained were analyzed by a statistician at the University of Leiden blinded to the identification of the groups of tissue biopsies collected. An average replicate value was calculated and log2 ratios were computed by comparing the matched pair biopsies and the gene expression differences for each transplanted kidney prior to and after reperfusion. One value per gene was calculated for the average expression of multiple probes with the same Entrez gene identification, resulting in 15,093 unique gene profiles. The unique gene profiles obtained from the whole genome array were analyzed by Ingenuity pathway analysis .In addition to biopsies collected for gene array studies, 20 transplanted kidneys were used to perform further biochemical analysis which included qPCR, western blot , and aldehyde enzymatic activity assays.μl that contained 15 μl of Fast SYBR© Green Master Mix (Life Technologies), 1000 nM primer  or 500 nM primer (ALDH4A1), and 10 ng cDNA. The cycling protocol was 20 seconds at 95°C, followed by 40 cycles of 3 seconds at 95°C and 30 seconds at 61°C. The melt curve protocol was 15 seconds at 95°C, followed by a minute at 60°C, followed by a gradual temperature increase from 60°C to 95°C (+0.03°C per 15 seconds) in 42 minutes.For qPCR, RNA was isolated from kidney biopsies taken at the end of the cold ischemic period. Kidney biopsy lysates were made by sonification, and RNA was isolated by the use of Ambion© RNAqueous Kit. To substantially reduce the possibility of DNA contamination in the preparations, the isolated total RNA was subject to precipitation with lithium chloride and DNase digestion (Ambion DNase-free). cDNA was made using the Takara© Primescript cDNA synthesis kit with oligo dT primers. qPCR reactions were performed in a final volume of 20 μg protein. Western blot was performed as described [Western blot analysis was used to quantify 4-HNE-induced protein adducts in addition to protein expression of ALDH enzymes. For western blot analysis, kidney biopsies taken 45 minutes after reperfusion were homogenized in mannitol-sucrose buffer  with protease and phosphatase inhibitors  using a glass homogenizer . Homogenates were subsequently collected and spun at 800 g × 10 minutes to remove nuclei and debris. The supernatant was collected and protein counts were obtained by Bradford assay and samples were normalized to escribed . Primaryμg of protein were used. ALDH enzyme activity was measured spectrophotometrically (340 nm) by analyzing the reaction of NAD+ to NADH as previously described [μmole NADH/min/mg of protein.To determine ALDH enzyme activity, 25 escribed , 12. TheP values for the change in expression comparing the paired biopsies, fitting into predefined pathways. P values are expressed as the –log P value. Results were then analyzed by biostatistical methods using average replicate values for each group of samples. Log2 ratios were computed and one value per gene was calculated for the average expression of probes with the same Entrez gene ID. For qPCR and protein assays, data was analyzed with GraphPad prism.Sample sizes were chosen for this study based on previous experience the University of Leiden has on conducting clinical studies regarding ischemia-reperfusion injury in human kidney transplantation , 14. SPSp-values using Ingenuity pathway analysis were calculated and initially reported [p-values in kidneys which did not develop delayed graft function compared to the kidneys which developed delayed graft function , aldo-keto reductases (AKR1A1), and glutathione-S-transferases  known to metabolize reactive aldehydes typically generated by the mitochondria during reperfusion . Togethe∗, ALDH7A1: 1739.9 ± 65.5 versus 1230.4 ± 83.1∗, ∗P = 0.001) with ALDH4A1 just at statistical significance  remained unchanged . Additionally, the amount of 4-hydroxynonenal-induced protein adducts in the reperfused kidneys was significantly higher for kidneys developing delayed graft function versus those that did not .Since the gene array findings suggest a link to reactive aldehyde metabolism, we further examined reactive aldehyde metabolism and aldehyde-induced protein adducts by obtaining paired biopsies from additional grafts that did not develop delayed graft function and donor kidney grafts that developed delayed graft function. For these biopsies, we determined the ALDH-dependent enzymatic activity to metabolize reactive aldehydes (using the substrate NAD+) in addition to 4-hydroxynonenal- (4-HNE-) induced protein adducts for biopsies taken 45 minutes after reperfusion. The total ALDH enzymatic activity was significantly higher for donor kidney biopsies without delayed graft function when compared to biopsies from kidneys that developed delayed graft function . As a comparison, ALDH1A1 was unchanged . Western blot for ALDH2 also showed a relative change in expression that did not reach statistical significance . No correlation between ALDH enzymatic activity and cold ischemia times for living donor grafts without delayed graft function was found, and the range of cold ischemia time for these kidneys was small . In addition, an inverse association also existed with ALDH enzymatic activity when compared to the length of hospital stay .However, ALDH enzymatic activity was also examined in relation to glomerular filtration rate (GFR) measured in patients at postoperative day 7 and length of hospital stay. Interestingly, those kidney biopsies that had lower ALDH enzymatic activity also had a lower GFR at day 7 , kidney injury molecule 1 (KIM-1), interleukin18 (IL-18), klotho, cystatin C, and liver type fatty acid binding protein (L-FABP) . RecentlAlthough associations exist between serum malondialdehyde levels and delayed graft function , 22, serin vitro, 4-HNE can inhibit human recombinant ALDH2 resulting in a decreased capability for the enzyme to metabolize reactive aldehydes [μM 4-HNE, most of the effect is reversible; however, at 500 μM 4-HNE concentrations, this effect is noted to be irreversible [Our data also examined ALDH7A1, ALDH4A1, and ALDH2 within the ALDH enzyme family suggesting that these enzymes are involved and contribute to a decreased reactive aldehyde metabolism seen in kidneys developing delayed graft function. However, the contribution of other families of enzymes such as AKR and GSTA cannot be excluded. Although protein expression was not significantly changed for ALDH2 in our study, it cannot be ruled out that ALDH2 was posttranslationally modified by reactive aldehydes. To identify posttranslational modifications, immunoprecipitation of an enzyme (such as ALDH2) followed by western blot for aldehyde-induced protein adducts could be performed. However, the amount of protein needed to conduct this type of study exceeded the amount obtained from a renal biopsy for our study. Based upon prior studies performed ldehydes . At 50 μversible . Since wversible , the rea∗2, which severely limits the metabolism of reactive aldehydes [∗2 variant as a predictor of delayed graft function, this may be due to organ transplantation (and in particular kidney transplantation) numbers that are traditionally low in East Asia compared to the rest of the world secondary to cultural reasons [In the era of precision medicine, it is also important to determine how genetic polymorphisms in mitochondrial ALDH enzymes may affect reactive aldehyde metabolism and delay graft function. In particular, ~560 million people in the world have a genetic variant of ALDH2, ALDH2ldehydes . Althoug reasons . Further reasons , 28. The reasons , 12.Our study does have a number of potential limitations that need to be considered when interpreting the data presented. Although we show an importance of reactive aldehyde production and metabolism in delayed graft function, we could not conclusively identify all the enzymes involved based on the size of the kidney biopsy obtained. Therefore, we only focused on 3 ALDH enzymes even though AKR and GSTA family members also had an increased gene expression for kidneys that did not develop delayed graft function. Additionally, the decreased protein expression observed might also be due to loss of cell and mitochondrial integrity since donor death itself induces metabolic dysregulation and mitochondrial dysfunction. We also compared grafts from living donor kidneys to deceased donor kidneys that developed delayed graft function. Further studies will be needed to compare reactive aldehyde production and metabolism also in deceased donor kidneys that did not develop delayed graft function as the effect we identify may be secondary to comparing living donor kidneys to deceased donor kidneys.The study is also an association study and will require further validation both in experimental models and in the clinical realm. Although we did find associations for aldehyde enzymatic activity with GFR and length of hospital stay, these associations should be considered with caution as many factors contribute to the postoperative recovery from a kidney transplant and a larger study would be important to confirm these findings.Together, we suggest that a decreased ability to metabolize reactive aldehydes in donor kidneys that develop delayed graft function occurs . This di


Purpose: The aim of this study was to prepare formulations of theophylline microbeads usingpregelatinized breadfruit starch  in combination withsodium alginate and chitosan at various polymer: drug ratios. Microbead formulations forcontrolled delivery of theophylline would be better alternatives to conventional dosage formsfor optimized drug therapy.Methods: The native and pregelatinized starches were characterized for morphology (scanningelectron microscope), crystallinity , thermal flow , density and flowproperties. Theophylline microbeads were prepared by ionic gelation and characterized usingsize, swelling index, entrapment efficiency and time required for 15% and 50% drug release (t15and t50 respectively).Results: FTIR and XRD spectra revealed the orderly arrangement of granules of the semi-crystallinebreadfruit starch was disrupted on gelatinization. The viscosity and flow of pregelatinized starchwere enhanced. Theophylline microbeads were near spherical in shape with size range 1.09± 0.672 to 1.58 ± 0.54 mm. FTIR and XRD spectra confirmed there was no drug-polymerinteraction. Microsphere size, swelling increased while entrapment and dissolution time (t50)reduced with polymer: drug ratio. The entrapment efficiency ranged from 30.99 ± 1.32 to 78.50± 2.37%. Optimized formulation, starch: alginate ratio 3:1 at polymer: drug ratio of 2:1, gave aprolonged dissolution time (t50 = 8.40 ± 1.20 h).Conclusion: Breadfruit starch was suitable as a copolymer for the controlled delivery oftheophylline in microbeads which could serve as a substitute to synthetic polymers in drugdelivery. Hydrogel beads are produced by introducing drug-loaded polymeric solution into the aqueous solution of polyvalent cations to produce a three-dimensional network of ionic crossed-linked moiety.14 When sodium alginate and chitosan are used in the ionic gelation method, the two polymers form the polyelectrolyte complex via the ionic interaction between the carboxyl residues of alginate and the amino residues of chitosan.15 Thus, cross-linking of alginate and chitosan in a hydrogel can be used to provide sustained release of drugs in which the resulting systems have enhanced stability compared to those obtained with a single polymer. In a previous study, slow release formulations of imazaquin were prepared by encapsulating the herbicide in starch and chitosan beads that were reinforced with alginate.16 The findings showed highly porous spherical beads, with the beads containing the starch-alginate-chitosan blend being larger and less porous than those containing only the chitosan-alginate blends. In another study, microparticles of stigmasterol were obtained using a blend of polymers of sodium alginate, starch and chitosan as the coating materials through a one-stage process using the external gelation technique. Resultant microparticles were spherical, with an encapsulation efficiency of 90.42% and method yield of 94.87%.17 The incorporation of starch to the chitosan-alginate blends in these instances appear to modify the porosity, size and release characteristics of the formulations.Microparticulate drug delivery systems are ideal for controlled delivery applications due to their biocompatibility as well as their ability to encapsulate a variety of drugs and sustain drug release.18 Theophylline has a half-life of about 8 hours. The immediate release formulations of theophylline are usually administered in 2-3 doses daily. The drug is rapidly and completely absorbed after oral administration with side effects including stomach/abdominal disorder, nausea and severe vomiting. In order to optimize drug therapy, reduce the dose and frequency of drug administration, and subsequently improve patient compliance, controlled release formulations are desirable. Thus, to achieve prolonged release of theophylline, microbead formulations could be better alternatives to the conventional dosage forms. The objective of this study is to formulate theophylline microbeads of theophylline using pregelatinized breadfruit starch as co-polymer with alginate and chitosan at varying polymer: drug ratio.The drug of choice in this study is theophylline ,which is commonly used in the treatment of respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma.Breadfruit was purchased from a local market in Owerri, Imo State, Nigeria. Theophylline was obtained from Wuhan Hezhong Bio-Chemical Manufacture Co., Ltd., Wuhan city, China. Sodium alginate was obtained from S.D. Fine Chem . Chitosan was obtained from Sigma-Aldrich, St Louis, MO, USA. Calcium chloride was obtained from Loba Chemie Pvt. Ltd., Mumbai, India.Mature, unripe breadfruits were washed, peeled and cut into small pieces which were soaked in distilled water. The resulting mixture was blended to obtain a slurry that was strained through muslin cloth followed by suspending the filtrate in distilled water to enable settling. The supernatant was decanted at 12-hour intervals and the starch slurry was re-suspended in distilled water. The starch cake was collected after 72 hours and dried in a hot air oven at 50°C for 48 hours. The dried mass was pulverized and then screened through a 250-µm sieve.To prepare the pregelatinized starch, an aqueous slurry of the dry native starch (100 g) was dispersed in 500 mL of distilled water and then heated at 100°C with constant stirring for 45 minutes. The resulting paste was dried in a hot air oven at 50°C for 48 hours and blended.The native and modified breadfruit starches were examined for shape and size using an optical microscope . (FT-IR) analysis  in transmission mode. The transmission spectra range was 4000 - 400 cm-1 using 64 scans with resolution of 8 cm-1.The starches were analysed by Fourier Transform-Infra Redo at a scan rate of 12°/min. The integration time was 0.150 seconds and step was 0.03 °.The X-ray diffraction (XRD) pattern was recorded using an X-ray diffractometer  with copper- cobalt radiation. The native and modified starch samples were exposed to the X-ray beam at 25°C. The scanning region of the diffraction angle (2ᶿ) was from 5° to 70 The starch sample (about 5 mg on a dry weight basis) was heated from 60 to 300°C in sealed aluminum pans at a scanning rate of 10°C /min. Thermal transitions were characterized by onset temperature, peak temperature, conclusion temperature and the gelatinization enthalpy (ΔH).Thermal characteristics of the starches were studied using a differential scanning colorimeter, DSC θ) was determined as:Ten grams of starch was weighed and poured through a funnel, into an opened cylinder placed on a wooden base of similar diameter. The starch was allowed to cascade into a heap. The angle of repose (Aqueous slurry of starch samples (10% w/v) were placed in 15 mL centrifuge tubes and allowed to stand for 5 min. The starch slurries were then centrifuged at 1000 rpm for 5 min and the supernatant was discarded. The sediment was weighed to determine the gain in weight of dry sample as the amount of water sorbed by the starches.pH of a 1% w/v aqueous solution of native and modified breadfruit starches were determined .19The bulk density of each excipient was determined by pouring 10 g of the powder at an angle of 45 through a funnel into a glass measuring cylinder with a volume of 50 ml. The bulk density was measured as the ratio of mass to volume occupied by powder. The tapped density was measured by applying 100 taps to 10 g of powder in a graduated cylinder. The solvent pycnometer method was used to determine the density of the starches using xylene as the non-solvent.The viscosity of a 1 %w/v aqueous slurry of each starch was determined using Brookfield rheometer  using CPE 40 and spindle sizes of 2 and 4 at 27 ± 2°C.All determinations were done in triplicates with the values of mean and standard deviation presented.Microbead formulation trials were carried out by varying the ratio of the pregelatinized breadfruit starch to sodium alginate, concentration of calcium chloride solution, amount of chitosan, ratio of total polymer to drug, stirring speeds and curing times. Optimized formulations obtained were prepared as described below:A 2% w/w aqueous solution of sodium alginate and pregelatinized starch blend was prepared . To the alginate-starch polymer blend was added 1 g of theophylline . Stirring was done continuously for 20 min using a magnetic stirrer. The drug-loaded polymer solution was manually extruded (using a syringe with needle size 21G) into 200 ml of calcium chloride (10 %w/w) that contained 0.2% w/v chitosan solution ( in 1%v/v dilute acetic acid) inside a 250-mL beaker. Stirring was continuous for 15 minutes at room temperature (25 ± 2°C); the beads formed were collected, washed with distilled water to eliminate the excess calcium ions and then dried overnight at 50°C. The composition of the prepared formulations was varied changing the ratio of starch: alginate (2:1) and polymer: drug (1:1) while formulations containing alginate alone were used as standard.The microencapsulation yield was determined by the ratio of the dry weight of the microbeads that were obtained to the total weight of solid raw materials (drug and polymers) used in the preparation of microbeads.The morphology and surface characteristics of the microbeads were observed using a scanning electron microscope, SEM  at an accelerating voltage of 25 kV.o to 70o at a scan rate of 12 ᵒ/min.The XRD pattern was recorded using an X-ray diffractometer  with copper- cobalt radiation. The scanning region of the diffraction angle (2ᶿ) was from 5) in transmission mode. Pellets of the samples were prepared by finely grounding with KBr under a hydraulic pressure of 600 dynes/m2. The spectra were scanned between 4000 to 400 cm-1 (32 scans with resolution of 8 cm-1).The drug, pregelatinized starch, alginate, chitosan and drug-loaded starch-alginate-chitosan microbeads were analysed by FT-IR  were accurately weighed, crushed and suspended in 100 mL of phosphate buffer pH 6.8  at 270 nm. The drug entrapment efficiency (E) was determined as follows:One hundred milligrams of microspheres were measured into a 100-mL volumetric flask. Water was added to the 100-mL mark and the microspheres were suspended in phosphate buffer pH 6.8, shaken vigorously and then left for 24 hours at room temperature while shaking intermittently. The supernatant obtained was centrifuged and the drug content was determined by UV spectroscopy using a UV spectrophotometer  at 270 nm.in vitro dissolution studies were carried out using the paddle method (USP XXXVI), rotated at 50 rpm in 900 mL of phosphate buffer pH 6.8 and maintained at 37 ± 0.5°C. Ten milliliters of dissolution medium were withdrawn at time intervals and replaced with equal amounts of fresh medium. The amount of theophylline released at each time interval was determined at a wavelength of 270 nm, using a UV spectrophotometer . Determinations were made in triplicate. The results of the drug release for the formulations was fitted to zero order, first order (ln Qt = ln Q0 + K1t), Higuchi (Q = KH√t), Hixon-Crowell (Q01/3– Qt1/3= K t) and Korsemeyer–Peppas (Qt /Q∞ = K tn) kinetic equations. The model of best fit was identified by comparing the values of the correlation coefficients.Microbeads containing 50 mg of theophylline were accurately weighed and suspended into 900 mL of phosphate buffer pH 6.8. The A statistical analysis was conducted to compare the effects of the polymers on the microbead properties using the analysis of variance (ANOVA). At a 95% confidence interval, probability, p values less than or equal to 0.05 were considered significant.20 In addition, this interaction produces a reduction in the porosity of the alginate beads thereby minimizing the leakage of the encapsulated drug. Further interaction occurs via intermolecular hydrogen bonding, similar to the mechanism of starch—alginate cross linking. The chitosan acts a coat that enhances the rigidity of the microbeads while reducing their permeability.In this study, the matrix of starch-alginate polymer blend was modified with chitosan. The mechanism of interaction between starch and alginate blend was intermolecular hydrogen bonding. The incorporation of starch provides a filling material that reduces the permeability of the otherwise porous alginate microbeads while aiding stability. The complexation that occurs between alginate and chitosan is via an ionic interaction between the carboxyl residues of the alginate and the amino terminals of chitosan.21 Photomicrographs of native and pregelatinized breadfruit starch are shown in -1 and is correlated with the XRD results which revealed disruption of the internal order of the starch granules in the pregelatinized form. DSC results showed endothermic peaks with the onset, peak, conclusion temperatures and enthalpy for the native breadfruit starch at 59.86°C, 142.81°C, 150.67°C and 1332.18 J/g respectively, whereas those for the pregelatinized starch were 61.60°C, 144.77°C, 152.58 °C and 1686.12 J/g respectively.The yield of starch obtained from breadfruits was 38.23%w/w. This appears to be a reasonably good yield considering the high water content of the fruit.22 During pregelatinization of starch granules, the amylose is precipitated and releases amylopectin which is responsible for swelling and water retention ability. When in contact with water, pregelatinized starch readily forms a gel and the strength of the gel is related to the amylose component. On the other hand, the cohesiveness of the gel depends on the amylopectin component.The material and physicochemical properties of native and breadfruit starches are presented in P < 0.05). The Carr’s index and Hausner’s ratio values were obtained from the bulk and tapped densities. The values revealed that modification of native breadfruit starch by pregelatinization improved the flowability of the powder but reduced its compressibility. This enhancement of flow in the pregelatinized starch was further confirmed using the angle of repose. The higher the angle of repose, the more cohesive the powder and the poorer the flow of the powder.The particle, bulk and tapped densities of the modified starch were significantly higher than those of the native starch  were near spherical in shape. Their size ranged from 1.09 ± 0.672 to 1.58 ± 0.54 mm as presented in 8 < B7 < B2 < B1 < B4 < B3 < B6 < B5. The microbeads containing breadfruit starch were larger than those containing alginate only (B7 and B8). The size appeared to increase with starch content. This could be due to the increased viscosity of the starch-alginate polymer blend with increased amounts of incorporated starch, leading to an increase in the amount of polymer-drug droplets extruded into the chelating medium, thus forming larger microbeads.The ionic gelation method is a simple, reproducible process devoid of the use of organic solvents and high temperatures. The yield of microbeads was 91.25% w/w. Scanning electron images (SEM) for the microbeads are shown in −1 is due to the hydroxyl group O−H stretching vibrations which is characteristic of all the natural polysaccharides. The strong asymmetric stretching absorption band at 1650 cm−1 and the weaker symmetric stretching band near 1430 cm−1 are due to the presence of carboxylate anions. The band at 1993 cm−1 is also assigned to asymmetric stretching of −COO− groups. The peak at 1058 cm−1 was shifted to lower wave-numbers in the microbead formulation. The bands observed at 1092 and 1043 c m−1 are characteristic of the C−O−C link. For the FTIR spectrum of chitosan, two weak peaks were observed between 2400 and 2350 cm−1 that were shifted to 2900 cm−1 in the microbead formulation. The characteristic medium intensity band at 4000 - 3500 cm−1 were also observed for chitosan. The FTIR spectrum of theophylline showed a broad and weak transmission band of N-H stretching vibration at 3000 cm-1. The N-H bending vibration was observed at 1663 cm-1 while stretching vibrations were observed at 1561 cm-1. The carbonyl group (C=O) was observed at 1705 cm-1 whereas the C-H stretching vibrations superimposed upon the N-H broad bend at 3054 and 2983 cm-1.The FTIR spectra of sodium alginate, chitosan, pregelatinized breadfruit starch, theophylline and the starch/alginate-based theophylline microbeads are shown in As revealed in the XRD pattern of theophylline in 8 < B7 < B2 < B1 < B4 < B3 < B6 < B5. Microbeads containing a starch to alginate ratio of 3:1 (Batches B5 and B6 ) had the highest swelling and this can be attributed to their high starch content. The entrapment efficiency values were within the range of 44.90 ± 6.20 to 78.50 ± 2.37%. The amount of drug entrapped increased with starch content and entrapment efficiency was in the same rank order as swelling. These results indicate that the presence of starch enhanced entrapment when compared to microbeads containing sodium alginate alone.The values of the swelling and entrapment efficiency of theophylline microbeads are also presented in 15 and t50 (time required for 15 and 50% drug release respectively) were determined and are also presented in 27 The release pattern of the theophylline microbead formulations showed that Batches B7 and B8  as well as B1, B4 and B6 had an initial burst release as observed from the short period of t15 (<60 minutes).29 This was followed by slow release of the drug. Microbeads with smaller sizes released a higher percentage of the drug which may be due to their larger surface areas. The presence of starch contributed to the delayed release of theophylline due to its matrix structure which reduced porosity and decreased the leakage of the encapsulated drug. Chitosan is also known to form a cross-link with sodium alginate, leading to blockage of pores on the surface of the microbeads, thereby sustaining drug release. Chitosan also enhanced the mechanical strength of microbeads.30 The ranking of the batches in terms of t50 was: B 5 > B3 > B1 > B6 > B4> B2> B7 > B8. The percentage of drug released decreased with increasing amount of starch in formulation and with increase in polymer: drug ratio. The optimum formulation was B5 containing starch: alginate ratio 3:1 and polymer: drug ratio of 2:1 which gave a controlled dissolution time t50 of 8.40 ± 1.20 hours and entrapment efficiency of 78.50 ± 2.37 % which were significantly (P < 0.05) higher than those of the formulations containing alginate alone.The dissolution profiles of the microbeads are presented in 31 The drug release kinetics of the batches were simulated using: zero order, first order, Higuchi,32 Hixson-Crowell,33 Korsmeyer-Pepppas  and n >0.89 to a super case II transport.34 From the values of the slopes, the drug release mechanism from Formulation B2 is considered to be super case II transport. On the other hand, Formulations B4 and B5 fitted the Hixson-Crowell kinetic model, which describes the release from a system which there is a change in the surface area and the diameter of particles.33 Formulations B6 and B8 fitted the first order kinetic model, implying that the release of theophylline was from a porous matrix.35Release of a solid drug from the microsphere matrix involves the simultaneous penetration of the surrounding liquid, the dissolution of the drug, and the leaching out of the drug through interstitial channels or pores. The kinetics of drug release is important due to their influence on drug bioavailability. The physicochemical properties of the drug and the polymer have been shown to govern the release of drug from formulations which could affect their release kinetics.-Pepppas .34 Drug in vivo studies to confirm the versatility of pregelatinized starch of breadfruit as a sustained release polymer in the preparation of microbead formulations of theophylline and other drugs. Future research could also focus on formulating and evaluating starch-based theophylline microbeads in the form of dry powders to produce aerosols intended for inhalation.The ionic gelation method was found to be efficient and reproducible for the formulation of theophylline microbeads when pregelatinized breadfruit starch was used as a co-polymer in different blend combinations with sodium alginate and chitosan and at different polymer to drug ratios. The drug release profile of the microbeads showed dependence on the amount of starch used and the polymer drug ratio. Further research may still be required using Not applicable.The authors declare that there is no conflict of interest.


Elaphe radiata. The experimental design required that the snakes take into account the limits of their bodies when choosing a suitable hole for penetration into the shelter. The experimental setup consisted of two compartments, a launch chamber and a shelter, separated by a partition with openings of different diameters. The diameters of the holes and/or their position in the partition were changeable. The subjects were 20 snakes divided into two groups, for one of which only the locations of the holes varied; for another, both the location of the holes and the limits of the body varied. The body was increased by feeding the snakes. In the course of the first three experimental series the snakes formed the skill of taking into account the body limits, which manifested in the reduced number of unsuccessful attempts to select holes too small for their bodies. During the fourth series, with the locations of holes randomized for each trial, the snakes demonstrated behavioral flexibility, significantly more often penetrating into the shelter from the first attempt irrespectively of the location of the suitable hole. We argue that these results demonstrate the body-awareness in snakes.Body awareness was studied experimentally in the rat snakes  One of the essential aspects of consciousness, no matter whether it is an animal or human, is self-awareness, i.e. the ability of the subject to separate self from the environment and oppose to it . This phIn classic experiments to identify the prerequisites of self-awareness a mirror test was used that reveals the ability to recognize one’s own reflection in a mirror. In the experiments by G. Gallup , chimpanIt should be noted that this method is subject to significant criticism. Firstly, it is difficult to apply it to animals in which visual modality is not a leading one  . SecondlIn connection with the growing criticism of this methodological approach, more and more authors are striving to offer alternative methods for identifying the ability of self-recognition. In particular, the phenomenon of recognizing own chemical traces on surrounding objects, the so-called “olfactory mirror”, is investigated ,17,18.Another direction of research in self-reflection is the study of the phenomenon of the scheme of the body. When orienting in the surrounding space for the implementation of locomotion and manipulation, it is necessary to take into account the physical characteristics of own body: Limits, volume, mass—and relate them to the physical characteristics of external objects. The body schema is a model of one’s own body as a whole, including also a set of ideas about the physical characteristics of one’s body  and its individual parts, allowing planning and carrying out various movements . AccordiMainly, modern authors are studying the features of the scheme of human body. Recent studies have shown that the body schema has high plasticity and is able to integrate external objects into its structure that are in physical contact with the individual—for example, the tools used by it ,27,28,29Elephas maximus) [Relatively recently, researchers began to focus on the body-awareness phenomenon—the ability of animals to take into account their own body’s relationship with environmental objects and to perceive their body as an obstacle to solving various problems. There is evidence of how different animals naturally take their bodies into account or their separate parts when overcoming obstacles . In the maximus)  these anmaximus) . Thus, iThe aim of our study was the phenomenon of body-awareness in snakes, or rather, a particular aspect of this phenomenon, expressed in their ability to take into account the limits of their own body when interacting with external objects. These animals were chosen as subjects for the reason that the task of penetration into various holes, e.g., to find shelter, is natural (eco-friendly) for them. In particular, after swallowing the prey, the borders of the body of the snake increase sharply, which affects the interaction with external objects. We propose that in connection with these characteristics of vital activity, they may have the ability to perceive their body as an obstacle to solving the problem of penetration into the shelter. Accordingly, in the experiment, we simulated a situation in which a snake, previously trained to enter a shelter through a certain hole, faced the impossibility of solving this problem in the same way when the experimenter changed the conditions in one of the two ways: Either by reducing the size of the hole, or by increasing the limits of the snake’s body (after feeding).Traditionally, reptiles are considered to have limited cognitive abilities in comparison with mammals and birds . HoweverThamnophis radix [Snake learning has been well studied in its various behavioral manifestations: Avoidance of stimulation (aversive stimulus), recognition of harmful and palatable substances , spatial orientation, conditioned aversion formation, conspecific recognition, habitat recognition, and cultural transmission of stimulus recognition ,43. In pis radix .Elaphe radiata. This is a rather large snake, up to 1.8 m long (adults). The body is slender, the head is small, poorly delimited from the body. The eyes are rather large. The color is light yellow, orange or bright brown. The snake inhabits various biotopes from plains to mountain forests, adheres to forest margins, often occurs along the banks of water pools, lakes, rivers, and in the fields of cultivated plants. Active during the daytime, feeds on rodents [Our subjects were snakes  rodents . In resp rodents . This asElaphe radiata: Ten males and ten females . All snakes were born and raised in the laboratory. Before the experiment, they were contained, one by one, in horizontal terrariums 700 × 400 × 500 mm with 12:12 h light:dark cycle. Soil: Pine bark-mulch, sphagnum, coconut crumb. Temperature in a warm corner was up to 31 °C, the background temperature 26–28 °C, overnight 20–24 °C; humidity 60%–80%. The animals were fed on rodents. All experimental snakes were naive.Twenty copperhead rat snakes The experimental setup comprised a glass terrarium with two compartments . Two ideThe experimental setup was located in an isolated room (with an area of 5 sq. m.) in order to exclude the effect of unaccounted variables. The compartments were separated by a glass partition, also painted black. In the partition there were three round holes with a diameter of 70 mm, located 5 mm above the floor. The diameter of the holes could be varied with the help of inserts placed on the inside of compartment B.Large hole (hereinafter L): D = 40 mm. The snakes could penetrate into this hole without difficulty even after swallowing food;Middle hole (M): Insert with a hole D = 10–14 mm (depending on the size of the snake). A snake penetrated through this hole without difficulty unless its body was enlarged by ingestion of food;Small hole (S) insert with hole D = 6–8 mm (depending on the size of the snake). The snakes could not penetrate into this hole.In the experiment, we used holes of three diameters:We emphasize that the inserts or holes in the partition were not supplied with any identification marks . Previous studies have shown that interaction with an unfamiliar environment causes an increased stress in reptiles, which affects the process of learning ,46; TherTo begin the trials, the snake was placed in a foam box with a plastic bottom and moved to compartment A of the experimental setup through the back cover. Then the plastic bottom was removed, leaving the snake on the ground. Next, we kept a time interval of 30 s before starting the trial, allowing the snake to stay in the box. The box was then lifted upwards, allowing the snake to move freely inside the experimental installation. Staying on a hard substrate in a bright room devoid of any shelters was a negative stimulation for the animal, and it tried to leave compartment A and hide in compartment B (the “wet” chamber) penetrating through one of the three holes . The triOnly one experimenter worked with the snakes. Latex gloves used to interact with animals and with substrate were changed after each trial. Initially, we set the maximum trial time to 10 min, after which the trial was considered unsuccessful and a snake would be removed from compartment A. However, during the experiment it was found that all the snakes solved the problem within a maximum of 4.5 min.Between the experimental trials within one series, a time interval of 10 min was maintained, during which the snake was kept undisturbed in compartment B (shelter). After each trial (while the snake was in compartment B), the partition between the compartments was wiped with soapy water and dried on the compartment A side to remove any possible odor trail. In addition, after each trial, the pebbles in compartment A were replaced with a new portion. In compartment B, the soil was mixed between trials when the snake was removed from this compartment through the back cover. To begin the next trial, the snake was placed again into compartment A using the previously described method.Note that usually experimental trials are carried out with a longer time interval than in our case (two times a day) ,47. HoweAn attempt was considered successful if the snake penetrated entirely into the compartment B through one of the holes;Unsuccessful was considered an attempt to penetrate the hole of type S, during which the snake pressed its muzzle to this hole, making wriggling body movements. In the case of type M hole, a penetration attempt was considered unsuccessful, during which a snake with enlarged body penetrated inside this hole to the expansion point of its body (the bulk produced with the swollen mouse), after which it retreated to compartment A. Partial insertion of the head into the hole was not considered an attempt. No snakes stuck in the holes.We registered the number of attempts to penetrate through different types of holes separately for each hole in each trial and separately for each snake in each trial. The penetration attempts were divided into successful and unsuccessful:The experiment was recorded on a (Sony HDR-CX405) video camera located above and behind compartment A at an angle of 45 degrees. The recording of each experimental trial was reviewed separately by three experienced experimenters to ensure an independent assessment and count of the number of penetration attempts. A penetration attempt was counted if all the three experimenters independently recorded its presence. Initially, we intended to solve controversial cases by consensus, but there were no such cases.The experiment consisted of 4 series . Each seExperimental group 1, when the size of the holes in the partition between compartments A and B was varied;Experimental group 2, when both the sizes of the holes in the partition between holes A and B and the borders of the body of snakes  were varied.The animals were divided in 2 groups, 10 individuals each:Series 1. The task was to form a skill in snakes of both samples to penetrate into compartment B through hole No. 1.Series 2. Conducted at the next bright phase following the end of series 1. In the interval between the end of series 1 and the beginning of series 2, the snakes stayed in the compartment B. The holes in the partition were closed. The inserts with the holes (according to the plan of the experiment) were installed immediately before the start of series 2. In group 1, the snake body limits remained unchanged, however, hole number 1 through which, in the previous series, they had formed the skill of penetration into compartment B, was made small (S), and hole No. 3 was made penetrable (M). Our task was to identify whether snakes, provided that the physical parameters of the body remained unchanged, but the environmental conditions changed, could find a new path to compartment B. In snakes of group 2 the limits of the body were increased by feeding the snake on a mouse of subadult age, due to which they were unable to penetrate entirely into the M-type opening, and, accordingly, could leave the launch chamber only through the L-type hole. Feeding was carried out in compartment No. 2, about 3 hours after the end of series 1. The swallowed food object was located in the stomach of a snake due to which the body section increased, making it impossible to penetrate into the holes of type M see . Our tasSeries 3 was carried out 3 hours after the end of series 2 and provided that by the end of series 2, the subjects of both groups managed to form a new penetration skill into compartment B through hole 3. In both groups, the permeable holes were moved to the position No. 2.Series 4 was conducted at the next bright phase after the end of series 3. The series consisted of 20 trials for each animal. In group 1 snakes, in each experimental trial, the holes were arranged randomly, provided that one of the holes has a diameter M and the others are S. We considered that snakes have the ability to take into account (be aware of) the natural limits of their body if throughout the entire series 4, they reliably make more successful attempts to penetrate the holes M. This would mean that the snakes are able to flexibly change their behavior, choosing a hole that is suitable in size to the borders of their bodies, irrespectively the position of the hole. In group 2 snakes, holes were also arranged randomly for each trial, provided that at least one of them was L, the others were M. Similarly, we believed that snakes have the ability to take into account (be aware of) the increased limits of their body, if during the entire series of 4 they reliably make more successful attempts to penetrate through the hole L.Statistical analysis was performed using Statistica version 12.6 . To identify the significance of reducing/increasing number of unsuccessful attempts to penetrate into various types of holes from the first to the last sample in each series, the Wilcoxon T-test was used. To identify the significance of the differences between the number of failed attempts in group 1 and group 2, we used the Mann—Whitney U test. To identify the significance of differences in the number of successful and unsuccessful penetrations into various types of holes, the Pearson’s chi-squared test was used. In the 4th series, the obtained distributions of penetration attempts into various types of holes were compared for a total of 20 samples with a uniform distribution.Our study involved noninvasive observations of animal behavior which were approved by Research Ethics Committee of the Institute of Psychology, Russian Academy of Sciences .In snakes of both groups during the 1st, 2nd and 3rd series there was a decrease in the number of unsuccessful penetrations into various types of holes from the first trial to the last. In all three series, all snakes reached the learning criterion see .The Wilcoxon signed-rank test was used to compare the number of unsuccessful penetrations into Compartment B  in the bp < 0.01). In series 3, such a significant shift was not identified, since already in the first trial, the number of unsuccessful penetration attempts was low . It should be noted that a decrease in the number of unsuccessful penetration attempts in the 1st trial of series 3 was found in snakes of group 1 in comparison with the 1st trial of series 1 . A similar pattern was found in group 2 . On the other hand, in group 2, the first trial of series 2 showed a significant increase in the number of unsuccessful penetration attempts in comparison with the first trial of series 1 . In addition, during the first trial of series 2, snakes from group 1 made significantly fewer unsuccessful attempts to penetrate into compartment B than the subjects of group 2 .It is interesting to note that at the end of the 2nd series, as well as in the subsequent series, the characteristic behavior was observed in the subjects of both groups, during which the snake crawled to different holes and drove its muzzle along the edges of the hole without making undulations in the direction of the latter. We believe that this behavior is indicative of the study by the animal of the size of holes based on the physical limits of its own body. Following this orienting behavior, the snake penetrated into one of the holes.During series 4 (with randomization of the location of the holes) there were no significant shifts in the number of unsuccessful penetration attempts in the subjects of both samples see . For theFor a statistical assessment of the prevalence of choices of penetrable holes in both samples, we used the Pearson’s chi-squared test. Based on the fact that for all 20 samples, 10 subjects of each sample made a total of 200 successful penetrations into compartment B, it turned out that in the 1st group this penetration was made from the first attempt in 178 cases and from the second attempt in 22 cases; in the 2nd group, successful penetration from the first attempt was made in 166 cases and from the second attempt in 34 cases. These empirical distributions were compared with theoretical distributions of random successful penetration into compartment B from the first attempt or after one or more unsuccessful attempts to penetrate into too small holes. The theoretical distribution was calculated on the basis that in each sample of 3 holes only one was penetrable for the body of the snake. Respectively, the probability of accidental penetration into compartment B from the first attempt was 33.3% (67 attempts). The results of the statistical analysis are shown in The data obtained for group 1, series 1–3, indicate the reduction of number of unsuccessful attempts to penetrate into compartment B through type S-holes. Changing location of the holes first leads to an increase in unsuccessful attempts (at the beginning of series 2), but then (in series 3) snakes steadily prefer to penetrate into the compartment B through the M-holes already during the first trials of the series. In subjects of group 1, by the end of series 1, the number of unsuccessful attempts to penetrate into the S-holes also decreases. However, at the beginning of series 2 , the number of unsuccessful attempts to penetrate through M-holes (previously penetrable) increases significantly . This may be because the experimental task in series 2 for snakes from group 1 was easier, they had only one variable changed, namely the size of the holes. In snakes of group 2 in the 2nd series, both the sizes of the holes and the borders of their bodies changed (the latter were enlarged). Meanwhile, the snakes of group 2 in series 3 already demonstrate a steady preference for penetration into compartment B through L-holes: from the beginning to the end of the series, the number of unsuccessful attempts to penetrate through the M-holes is reduced slightly.Accordingly, in the course of series 1–3, the snakes learned to penetrate into an outgrowth located in a certain part of the experimental setup (since the arrangement of the holes did not change during the series). However, by the end of series 3, the snakes of both samples had learned to recognize holes S and holes M, respectively, as impenetrable. This is evidenced, firstly, by the fact that already in series 3 from the first trial to the last there is no significant reduction in the number of unhurried attempts . Secondly, during series 4 , the snakes of both groups were significantly more likely to penetrate into compartment B from the first attempt regardless of the location of the permeable hole.Thus, the snakes of both groups exhibit behavioral flexibility. They transfer experience from one experimental series to another and begin to recognize specific hole sizes as penetrable to their bodies. For group 1  M-holes are penetrable. For group 2 with the body borders enlarged after feeding, the L-holes are permeable. In series 4, with a random arrangement of holes in each trial, snakes from both groups significantly more readily (as compared with random choice) choose a permeable hole  from the very first attempt, which also suggests behavioral flexibility.Elaphe guttata guttata), based on visual landmarks, could rapidly learn the position of a hidden goal in an open field task. The fact that in our study, snakes, before penetrating into the hole, drove the muzzle along the edges of this hole, suggests that the tactile sensations served as landmarks for them. In other words, they kinesthetically correlated the size of their bodies with the size of the hole. However, this finding needs additional experimental verification.Behavioral flexibility is the ability to adjust behavior to changes in the environment , by direMeanwhile, the results of series 4 (when the location of the holes changed for each sample) suggest that snakes may develop awareness of their body limits and compare them with the size of the holes, which is the basis for behavioral flexibility in choosing a hole to penetrate and hide in the shelter. In our opinion, the awareness of the limits of the body was formed during the first 3 series of the experiment. Thus, in our experimental situation, awareness of own body in snakes is the result of learning in the process of a number of trial and error attempts. The snakes from group 1 formed the skill of taking into account the natural limits of their bodies, whereas the snakes from group 2 formed the skill of accounting for increased (due to feeding) limits of their own bodies.In natural conditions, many snakes die, or are stuck, when encountering a hole they cannot penetrate, most probably exactly because the “learning of self-awareness” (or “taking into account the enlarged body”) requires a considerable number of trials, that is, a sufficient volume of prior experience. At least, this is one plausible explanation.Unfortunately, the studies aimed at the awareness of own body limits in various animal species are not numerous. In similar studies conducted on skinks, it was found that these animals are also able to take into account both natural and artificially increased (using objects attached to their heads) limits of their body when choosing a suitable hole to reach the lure . AmphibiPeriplaneta americana [In earlier studies carried out on cockroaches mericana , as wellThe results of a similar experiment on brown rats (Long-Evans)  suggest On the other hand, it is rather difficult to compare the data obtained in our experiment with the body-awareness of elephants  due to sStill we believe that the methodical approach to research in body-awareness proposed in this article may be effective for use on various types of vertebrates. Our method differs significantly from the method of self-recognition in the mirror ,13,14,15Reptiles are a key class in developing an understanding of the evolution of cognitive abilities among amniotes. The study of similarities and differences in their cognitive processes may provide new information on the homologies and analogies of cognitive mechanisms in amniotes in general . The datElaphe radiata are able to take into account both the natural limits of their bodies, and those enlarged by feeding, which makes them exhibit behavioral flexibility when choosing a suitable opening to enter the shelter.The results of the experimental study suggest that the rat snakes


Angiomyofibroblastoma (AMFB) is a benign mesenchymal tumor most commonly found in the female genital tract of premenopausal women. Although rare, AMFB is an important consideration in the differential diagnosis of vulvar and vaginal masses, as it must be distinguished from aggressive angiomyxoma (AA), a locally recurrent, invasive, and damaging tumor with similar clinical and pathologic findings.We describe a patient with a 4 cm vaginal AMFB and the relevant preoperative radiographic imaging findings.Preoperative diagnosis of AMFB remains difficult. Common findings on magnetic resonance imaging and transvaginal sonography are described. We conclude that both transvaginal ultrasound and MRI are potentially useful imaging modalities in the preoperative assessment of vulvar and vaginal AMFB, with more data needed to determine superiority of one modality over the other. Angiomyofibroblastoma (AMFB) is a rare, indolent mesenchymal tumor that most commonly occurs in the female genital tract in premenopausal women, most frequently in the vulva and vagina. AMFB typically measures less than 5 cm; however, case reports describe tumors of up to 23 cm in size . Few casAMFB was first described in the literature by Fletcher et al. in 1992 as an important distinction from aggressive angiomyxoma (AA), which is an infiltrative myxedematous mesenchymal tumor with the potential for local recurrence .Preoperative diagnosis of AMFB and distinction from other soft tissue tumors is often difficult, as there is limited information on characteristic imaging findings. The differential diagnosis for a vaginal or vulvar mass includes Bartholin's gland cyst, epidermal inclusion cyst, Gartner's duct cyst, fibroma, lipoma, hemangioma, leiomyoma, and alternative rare mesenchymal tumors. Here, we describe a case report of a vaginal AMFB and relevant radiologic features to aid in diagnosis and distinction from aggressive angiomyxoma.A 30-year-old gravida 1 para 1 female presented to our Emergency Department complaining of a vaginal mass present since the birth of her child 4 years earlier. At that time, she underwent an uncomplicated vacuum-assisted vaginal delivery and was unaware of significant lacerations or repairs. She felt that the mass had not changed significantly in size since the postpartum period, but she had never been evaluated by a physician. She complained of acutely worsening discharge over the previous month, described as watery yellow to pink and occasionally blood tinged. She denied changes in bowel movements, dysuria, hematuria, fevers, chills, night sweats, changes in appetite, or weight loss. She complained of both entry and deep dyspareunia.Physical exam was notable for copious serosanguinous fluid within the vaginal vault. A well-circumscribed, smooth cystic structure approximately 4 cm in diameter was noted along the posterior vaginal wall. There was also a 0.5x1.0 cm exophytic lesion overlying the mass with serosanguinous drainage . On rectTissue biopsies were taken; however, they were of limited diagnostic value, showing fibrous tissue with acute and chronic inflammation and squamous debris, consistent with cyst wall and contents.A pelvic ultrasound (US) was performed and showed a complex vaginal mass, inseparable from the cervix, measuring 5.1 x 3.8 x 5.4 cm . Color Dα-SMA), and progesterone receptor but demonstrated focal estrogen receptor positivity.The patient was taken to the operating room for an uncomplicated surgical excision. Histology was notable for hypocellular edematous myxoid stromal tissue alternating with hypercellular areas of stromal cells clustered around thin walled small to medium-sized vessels, consistent with angiomyofibroblastoma. Stromal cells were noted to be spindled with eosinophilic cytoplasm. Nuclei were round or ovular with fine chromatin . Rare miAlthough AMFB is a rare diagnosis, it is an important consideration in the premenopausal and perimenopausal patient presenting with a vulvovaginal mass given its predilection for this region of the female genital tract. In a review of the literature in 2015 by Wolf et al., 125 cases of female AMFB had been previously reported, of which 92% were either vulvar N = 98) or vaginal in origin N = 17). Median age was 45, and the majority of cases were observed in women less than 60 years of age [ or vagin. Median Differential diagnosis for AMFB includes Gartner's duct cyst, epidermal inclusion cyst, leiomyoma, and fibroepithelial polyps among the more common etiologies. It is most important to distinguish AMFB from aggressive angiomyxoma (AA), which can commonly be misdiagnosed based on anatomic and pathologic similarities . AlthougDiagnostic utility of preoperative imaging for AMFB remains controversial. However, as the number of reported cases increases in the literature, more data exists on common radiographic features .US is typically the first imaging modality used to evaluate vaginal masses due to its intrinsic high resolution, availability, and cost effectiveness. US can be utilized to distinguish vulvar and vaginal AMFB from AA and other mesenchymal tumors , 21. AMFIn contrast, US appearance of AA is typically a hypoechoic mass with homogenous echogenicity and occasional echogenic septa correlating to fibrous bands on pathology , 25. TheTo complete the differential diagnosis, vaginal wall cysts are anechoic masses without internal vascularity; vaginal epidermoid cysts are homogeneous hyperechoic masses without internal vascularity; and heterogeneous solid masses are often malignancies or leiomyoma .On MRI, AMFB is typically well circumscribed . It is rContrast enhanced imaging was only reported in 5 studies. Four showed homogenous hyperenhancement and one demonstrated heterogeneous enhancement . HyperenOur case demonstrated heterogeneously high T2 and low T1 signaling intensity on MRI which is consistent with the literature. Postcontrast sequences demonstrated enhancement of the wall, absent internal enhancement superiorly, and bulky nodular enhancement inferiorly, consistent with a complex cystic mass.When correlated histologically, hyperintensity on T2W and absent enhancement correspond to hypocellular areas with abundant collagenous stroma. Areas of hyperenhancement correspond to areas of hypercellularity and vascularity, with little collagenous stroma and water content. Intermediate intensity on T2W and less avid enhancement are thought to represent areas of intermediate cellularity and collagenous stroma .Data on MRI characteristics of AA is limited. AA has similar findings to AMFB on T1W and T2W images but is thought to be differentiated best by its enhancement characteristics. AA is described as having an intense, swirled or layered pattern. Similar swirling and layered enhancement is also described on contrast enhanced CT , 33–36. In conclusion, in the patient with a vaginal or vulvar mass, it is important to consider AMFB among the differential diagnoses, particularly because of the importance of distinguishing AMFB from AA for treatment purposes. Postoperative histologic examination is needed for definitive diagnosis; however, we propose that ultrasound and MRI are increasingly useful diagnostic tools, particularly as the number of reported cases increases. Given the cost effectiveness of ultrasound, initiating imaging workup of a solid vaginal or vulvar mass with ultrasonography is suggested.


By immunoblot analysis, antibodies to the NC16A domain and other extracellular domains (ECDs) of COL17 were detected earlier than antibodies to intracellular domains (ICDs) in the active BP model. Time course analysis by enzyme-linked immunosorbent assay demonstrated a delayed peak of antibodies to ICD epitopes in active BP model. The blockade of CD40–CD40 ligand interaction soon after the adoptive transfer suppressed the production of antibodies to the non-collagenous 16A (NC16A) domain but not to an ICD epitope, suggesting the sequential activation from T and B cells against the ECD epitopes including the NC16A domain to those against ICD epitopes in vivo. Both wild-type mice immunized with a fragment of the NC16A domain and the recipients of those spleen cells produced IgG antibodies to ICD and ECD epitopes, showing intramolecular epitope spreading from the NC16A domain to other epitopes of COL17. Furthermore, we found that a portion of the active BP model mice show intermolecular epitope spreading from human COL17 to murine BP230. The appearance of antibodies to ICD epitopes of COL17 or of antibodies to murine BP230 did not correlate with the skin changes in the mice, suggesting that those antibodies have low pathogenicity. These results suggest that the immune response to the ECD epitopes of COL17, especially to the NC16A domain, triggers intramolecular, and intermolecular epitope spreading to ICD epitopes of COL17 and to murine BP230. These novel findings provide insight into the mechanism of epitope spreading in organ-specific, antibody-mediated autoimmune disorders.Bullous pemphigoid (BP), the most common autoimmune blistering disease, is induced by autoantibodies to type XVII collagen (COL17). Previous studies demonstrated that COL17 harbors several epitopes targeted by autoreactive T and B cells and that the target epitopes change sequentially during the disease course. To elucidate the details of the humoral immune response to COL17, we used an active BP mouse model in which BP is induced by the adoptive transfer of spleen cells from wild-type mice immunized with human COL17-expressing skin grafting to immunodeficient COL17-humanized (Rag-2 BP characteristically affects the elderly, and recent studies reported a trend of increased incidence of BP . BP is rtibodies , 16. Thutibodies , 18. A ptibodies . Autoanttibodies . Furthertibodies .BP230 is another autoantigen of BP and was originally identified as the major antigen of BP , 22. BP2In vivo experiments using a human COL17-expressing skin-grafted BP mouse model showed that IgG antibodies to human COL17 initially react to the ECD epitopes and that, subsequently, the humoral immune responses target additional ECD and ICD epitopes  or to other proteins in the same tissue (intermolecular epitope spreading) over time , 30. Intepitopes . A prospepitopes . Thus, eRag-2−/−/COL17-humanized  mice. This model continuously produces IgG antibodies to human COL17 in a CD4+ T-cell-dependent manner and reproduces the BP disease phenotype. By using this active BP model, the current study demonstrates that the production of antibodies to the ECD epitopes of COL17 precedes that to the ICD epitopes, especially to the inner portion of the ICD. The interference of T- and B-cell interaction by monoclonal antibody to the CD40 ligand (CD40L) shows that T- and B-cell interactions for ECD epitopes precede those to ICD epitopes of COL17 in an active BP model. Wild-type mice that were immunized with a fragment of the NC16A domain produced antibodies to ICD epitopes of COL17. Furthermore, the active BP model generates antibodies to murine BP230 as a result of intermolecular epitope spreading. These findings clarify the details of epitope spreading in BP.To address these issues, we utilized an active disease model for BP that we previously established . It is gRag-2−/− mice were received as a gift from the Central Institute for Experimental Animals . We crossed COL17m−/−, h+ (COL17-humanized) mice that we had previously generated  mice.C57BL/6-background enerated  with Rag1 to Ser204), ICD-2 (Thr197 to Lys466), NC16A (Glu490 to Arg566), ECD-1 (Gly567 to Gly860), ECD-2 (Leu853 to Gly1218), and ECD-3 (Ser1211 to Pro1497) were generated as GST-fusion proteins using the expression vector pGEX6P-1  and the bacteria BL21 . These GST-fusion proteins were purified using GSTrap HP  according to the manufacturer's instructions. NC16A-R7 (Asp522 to Gln545), 2 ICD peptides  and 3 ECD peptides  of human COL17 were chemically synthesized . The amino acid numbering system is based on the human COL17 sequence (NP_000494.3). Recombinant proteins covering all parts of murine BP230 were generated as previously reported (1-Gly3264), containing the N-terminal (plakin) domain; BP230-2 (Glu3223-Pro5562), containing the (coiled-coil) rod domain; and BP230-3 (Asp5533-Gln7833), containing the C-terminal (intermediate filament-binding) domain, according to GenBank (AF396877.1) and a previous report  analysis, as described below. Spleen cells were isolated 5 weeks after the skin graft and pooled from several immunized wild-type mice, and 2.0 × 108 cells/mouse were adoptively transferred into Rag-2−/−/COL17-humanized mice through a tail vain in 500 μL of PBS as previously reported  at day 0 soon after the adoptive transfer of immunized splenocytes as previously reported . Serum samples were also obtained from recipient mice weekly and assayed by indirect IF microscopy and enzyme-linked immunosorbent assay (ELISA).Indirect IF using mice sera was performed on normal human skin (NHS) and wild-type mouse skin using standard protocols. We used 1:20 diluted mouse sera as the primary antibodies and 1:100 diluted FITC-conjugated antibodies to murine IgG  as the secondary antibodies.2O2. For the detection of antibodies to murine BP230, recombinant murine BP230 proteins were used as a substrate. The membranes were incubated with 1:20 diluted mouse serum samples overnight at 4°C. Bound antibodies were visualized using 1:5,000 diluted HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch). As a positive control, 1:5,000 HRP-conjugated mouse anti-His-tag mAb-HRP-DirecT  was used. The blots were detected using the ECL Plus Detection Kit .Immunoblotting of recombinant proteins covering human COL17 was performed as described previously . Briefly450 of tested serum—OD450 of negative control)/(OD450 of positive control—OD450 of negative control) × 100 (450 of tested serum—OD450 of negative control)/(OD450 of positive control—OD450 of negative control) × 100.To determine the titer of antibodies to the NC16A domain of human COL17 in serum samples from the experimental mice, 96-well microtiter plates coated with recombinant NC16A protein purchased from MBL were incubated with diluted mouse sera for 1 h at room temperature. After being washed, bound antibodies were developed with 1:40,000-diluted, HRP-conjugated antibodies to mouse IgG (Jackson ImmunoResearch Laboratories), and the OD was read at 450 nm. The ELISA index value was defined by the following formula: index = (ODl) × 100 . To examt-test with Welch's correction was used for comparisons of ELISA index in wild-type mice grafted with human COL17-expressing skin (skin-grafted wild-type mice). P-values of <0.05 were considered significant compared with the control.Data were expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism . The unpaired Rag−/−/COL17-humanized mice (n = 4)  . Becausereported , around observed . The dehobserved . Dermal-observed . The affobserved .Indirect IF examination using sera from the active BP model revealed that IgG bound to the DEJ of normal human skin. Time-course analysis revealed that IgG antibodies to the DEJ, which reflects the presence of antibodies to human COL17, became detectable in recipients' sera 1 week after the transfer. Although the titer gradually decreased after the peak, it remained high  for 10 weeks . We nextTo examine the presence of antibodies to various regions of human COL17 in the active BP model, we utilized 6 fragments of COL17 which were previously reported  36). By. By36). 522 to Gln545) peptide because we previously reported that IgG antibodies to R7, but not to other portions of the NC16A domain, are pathogenic in vivo  and 3 ECD peptides  with reference to previous studies (n = 16) at 5 weeks after the skin grafting and compared them to those in untreated wild-type mice (n = 8) by ELISA. Although we expected the skin grafting to induce antibodies to every epitope of human COL17, it preferentially induced antibodies to NC16A-R7 but not to ICD-149 nor to ECD-1330 (n = 6). As expected, the titer of antibodies to NC16A-R7 peaked at day 9 and decreased rapidly thereafter  and compared the titers of antibodies in untreated active BP model (n = 9)  at day 0 of the adoptive transfer ( (n = 9) . As we p (n = 9) , a singl (n = 9) . Antibod (n = 9) . Interes (n = 9) . These rRag-2−/−/COL17-humanized mice at 5 weeks after immunization (n = 8) (Rag-2−/−/COL17-humanized recipients (n = 8). As expected, the recipients receiving NC16A-R7-immunized spleen cells developed antibodies to NC16A-R7 shortly after adoptive transfer, and then the titer gradually decreased  . As a coecreased . Additioecreased , suggestn = 8), and they showed no reactivity (data not shown). We then examined sera from the active BP model at days 8, 21 and 84 (n = 8). No reactivity was observed in sera at day 8 (data not shown) nor at day 84  other than ICD-149 (Ala149 to Ser172). Second, a correlation between disease severity and epitope spreading remains unclear. As mentioned above, previous studies demonstrated that the disease severities of BP are associated with the appearance of antibodies to ICD epitopes in both mouse models and human patients . This is probably due to the lower antigenicity of those proteins compared to that of the NC16A domain. Fourth, we have not examined intermolecular epitope spreading to hemidesmosomal proteins such as plectin, integrin α6, integrin β4, or laminin 332, which were reported to interact directly with COL17  were higher at day 56 than at day 8, as shown in patients , 34. Meath COL17 –54, althin vivo, and it is dependent on the target epitope. These novel findings elucidate certain details of epitope spreading in BP and give us a hint for new therapeutic strategies for BP that involve the regulation of this phenomenon.In conclusion, the immune reaction to human COL17 that starts from ECD epitopes, especially those of the NC16A domain, was found to spread over time to ICD epitopes and to murine BP230 in an experimental BP model. The timing of the interaction between COL17-reactive T and B cells differs This study was carried out in accordance with the recommendations of the local ethics committee and the Institutional Review Board of Hokkaido University, with written informed consent obtained from all subjects in accordance with the Declaration of Helsinki. All animal procedures were conducted according to guidelines provided by the Hokkaido University Institutional Animal Care and Use Committee under an approved protocol.HU and NY performed the experiments. KN, KM, HI, and WN provided materials. HU and HS designed the experiments. HU wrote the manuscript, and all the coauthors reviewed the manuscript and gave final approval of the submission.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.