Proteomic analysis of early phosphorylated proteins in acute pancreatitis model

Background and Objective: The exact mechanism of acute pancreatitis (AP), which is an inflammation of the pancreas, still remains unclear. In this study, we examined the protein phosphorylation changes during the early stage of AP in mice using proteomic analysis. Methods: AP model in mice was constructed using an intraperitoneal injection of cerulein. Blood samples and pancreas were collected at 1, 3, 6, 9h after the final injection (n=3 at each time point). Samples collected 3h after the final injection were separately mixed and named S (saline group) and C1 (cerulein group); samples collected 6h after the final injection from the cerulein group were mixed and named C2. Proteins from S, C1, and C2 were extracted, digested by trypsin, and subjected to LC-MS/MS analysis, bioinformatics analysis, and Western blotting. Results: A total of 549 sites (426 proteins) were upregulated, and 501 sites (367 proteins) were downregulated in C1 compared to S; while 491 phosphorylation sites (377 proteins) were upregulated and 367 sites (274 proteins) were downregulated in C2 compared to S. Motif analysis showed that proline-directed kinase and basophilic kinase had a key role during early AP. During an early AP stage, the cellular distributions of proteins slightly changed. The types of domains changed with the development of AP. Phosphorylation proteins associated with calcium signaling, especially IP3R mediated calcium release, lysosome and autophagosome pathway, pancreatic digestive activation, and secretion, were found to be involved in the development of early AP independent of NF-kB activation. Moreover, the MAPK family was found to have a greater impact at the early stage of AP. We also found differentially expressed phosphorylations of amylase and trypsinogen and increased phosphorylation of MAPK6 S189 in early AP. Conclusion: IP3R mediated calcium release and activation of MAPK family are key events promoting the development of early AP.

Subcutaneous Tocilizumab for Thyroid Eye Disease

Purpose: To demonstrate the efficacy of subcutaneous tocilizumab (TCZ-SC) treatment for recalcitrant thyroid eye disease (TED) refractory to intravenous methylprednisolone (MP) and oral methotrexate (MTX).Case summary: (Case 1) A 52-year-old man, smoker, with hyperthyroidism presented with a 3-months history of TED. The initial clinical activity score (CAS) was 5 (total score of 7). High-dose intravenous MP and oral MTX failed to improve the symptoms. He was treated with four doses of 162 mg TCZ-SC at an interval of 2 weeks. Pre-treatment laboratory test results were within normal limits. CAS was reduced to 2 weeks after the final injection, and there were no recurrences during the 6-month follow-up. (Case 2) A 37-year-old woman, non-smoker, with a 3-months history of hyperthyroidism presented with conjunctival injection and upper eyelid erythema. The initial CAS was 4, and thyroid-stimulating immunoglobulin level was raised (475% of normal). The symptoms did not improve with 7.5 g intravenous MP and oral MTX. The patient was treated with 162 mg TCZ-SC. Pre-treatment laboratory test results were within normal limits. CAS was reduced to 1 after the final injection, and there were no recurrences during the 6-months follow-up.Conclusions: TCZ-SC improves symptoms of corticosteroid-resistant TED, and may be a reasonable option in recalcitrant TED cases. However, further studies are required to justify the use of TCZ-SC for TED.

Botulinum toxin injection without electromyographic guidance in consecutive esotropia

Purpose To investigate the efficacy of botulinum toxin injection without electromyographic guidance for the treatment of consecutive esotropia. Methods A retrospective study was performed on 49 subjects with consecutive esotropia who received botulinum toxin injection in the medial rectus muscles without the use of electromyographic guidance. Treatment was considered successful if the final ocular alignment was orthotropic or esodeviation was ≤10 prism diopters (PD) during distant fixation. Results The mean age was 15.2 ± 8.3 years. The mean esodeviation before injection was 21.8 ± 9.1 PD at distance and 21.3 ± 8.3 PD at near. The mean number of injections per patient was 1.3 ± 0.7, and 46 patients (93.9%) received two or fewer injections. At 6 months after the final injection, the mean angle of esodeviation was 7.3 ± 6.0 PD at distance and 7.5 ± 6.6 PD at near (all p<0.001), and 69.4% showed successful alignment. By multivariate analysis, an initial postoperative esodeviation of ≤18 PD at one month after exotropia surgery was considered to be a predictive factor for successful botulinum toxin injection (P = 0.007). Vertical deviation and/or ptosis occurred in 4 patients (8.2%) at two weeks after injection, which all resolved within three months. There was no recurrence of exotropia up to the final follow-up examination. Conclusion Botulinum toxin injection without electromyographic guidance is safe and effective in the treatment of consecutive esotropia without causing recurrent exotropia. Successful botulinum toxin injection is likely in patients with an initial postoperative esodeviation of 18PD or less at one month after exotropia surgery.

Analysis of Cell Fraction of 99mTc-HMPAO Radiolabeled Leukocytes

Purpose: 99mTc-HMPAO radiolabeled autologous leukocyte scintigraphy is routinely used clinically for infection imaging. Leukocytes are mostly separated via sedimentation. It is unknown whether leukocytes are clearly separated by sedimentation or selectively labeled. Therefore, in this study, the blood cell numbers were investigated after leukocyte radiolabeling to identify the cells strongly radiolabeled by 99mTc-HMPAO. Methods: This study was performed with leftover blood samples of the patients who underwent 99mTc-HMPAO scintigraphy at Chonbuk National University Hospital (2018-2019). The blood of 22 patients was drawn for 99mTc-HMPAO scintigraphy. WBCs were separated via conventional sedimentation at our clinic and radiolabeled. The concentration of cell components was determined using an automatic hematology analyzer. The cells in the final sample injectate sample were separated using Histopaque and counted with a dose calibrator. Results: The average numbers of RBCs, WBCs, and PLTs in the final injection sample were 79 ± 33, 23.26 ± 11.95, and 229.5 ± 206.57 x 103/μL, respectively. The PLT number was almost 10-fold the number of WBCs. The number of RBCs was nearly 3-fold higher than WBCs [RBC/WBC ratio = 4.67 ± 3.58, and PLT/WBC ratio = 10.65 ± 12.46]. Following Histopaque separation, the activity of each layer showed 99mTc-HMPAO labeling of WBC > RBC > PLT in order. The total activity/cell numbers of WBCs, RBCs and PLTs were 0.016 ± 0.010, 0.005 ± 0.005 and 0.003 ± 0.002, respectively (p > 0.05). Conclusion: Although the numbers of RBCs and PLTs were highly increased after sedimentation, their individual cellular activity was lower than that of WBCs. 99mTc-HMPAO was more selective to WBCs than RBCs or PLTs. In conclusion, a higher number of WBCs were radiolabeled compared with RBCs and PLTs.

Principles of Injectable Facial Filling

Currently, the term “fillers” refers to injectable off-the-shelf products used to treat skin irregularities and volume deficiencies. The use of fillers to correct wrinkles and creases as well as to restore volume to the aged face has grown rapidly. The clinician must fill lines and creases as well as undertake the more difficult task of creating three-dimensional shapes in the subcutaneous space with a two-dimensional instrument. Local anesthetic temporarily adds facial volume in order to demonstrate the intended result of the final injection. This technique is invaluable because “previewing” the intended result allows the clinician and client to make adjustments with impunity.

OvSynch program, its modifications and alternative hormonal programs in cow reproduction

The OvSynch program was formulated over 20 years ago (it consists in administering gonadoliberin on the day of starting the procedure, then injecting prostaglandin 7 days after the GnRH initial injection, and administration of GnRH two days after applying PGF2α. The procedure ends in the insemination of a female 16–20 hours after administering the final dose of the hormone) and it is commonly applied in cow reproduction management protocols. It has evolved into numerous modifications which has boosted its effectiveness. They have consisted in replacing GnRH with hCG or pLH, diversifying the time of the final injection of GnRH, which follows the prostaglandin injection, and postponing insemination after the final GnRH dose, as well as changing the dosage of the aforementioned hormones. To expedite the Ovsynch protocol, so-called PreSynch was used, which involved administering two prostaglandin injections with a 14 day interval before applying the initial GnRH dose in protocol OvSynch. The interval between the second prostaglandin injection in the PreSynch program varied (PreSynch-14, PreSynch-12, PreSynch-11, PreSynch-11 and PreSynch). The pregnancy rate can be also improved by introducing GnRH injection before starting the OvSynch program, administered 7, 6 or 4 days before the initial GnRH injection in OvSynch (G7G, G6G, G4G). Higher pregnancy rates, in comparison to the PreSynch-OvSynch program, can be obtained by applying the DoubleOvsynch protocol. In this protocol two OvSynch programs are administered in a 7-day interval. Lately, alternative programs such as DoubleSynch and EstraDoubleSynch have emerged. The DoubleSynch protocol consists in administering PGF2α in day 0, GnRH on the second day, another PGF application on the 9th day, and the second GnRH dose on the 11th day. Artificial insemination is performed in oriented time (TAI). This solution is used in order to achieve a higher percentage of pregnant cows in comparison to the PreSynch+OvSynch protocol. It also enables synchronisation of heat and ovulation, both after the first and the second hormonal treatment of GnRH. In EstraDoubleSynch protocol the second injection of GnRH at the end of the DoubleSynch program is replaced by estradiol benzoate (BE) administration on the 10th day, in order to acquire a higher level of follicle waves synchronisation and ovulation rate. Those programs applied in cow reproduction management can positively influence fertility and boost cow breeding profitability..

Optimal Solubility of Diclofenacβ-Cyclodextrin in Combination with Local Anaesthetics for Mesotherapy Applications

Because of low injection volume, the recently marketed injectable solution of diclofenac in complex withβ-cyclodextrin (Akis®, IBSA Farmaceutici Italia) is an ideal candidate for mesotherapy applications. In this study, we investigated the solubility of Akis, 25 and 50 mg/kg, in combination with various local anaesthetics (lidocaine, mepivacaine, bupivacaine, levobupivacaine, and ropivacaine) at different concentrations in aqueous vehicles (normal saline, sterile water, or bicarbonate). Final injection mixtures were classified as limpid, turbid, or milky at visual analysis under standardized conditions. We found that (i) the use of sterile water for injections or normal saline as vehicles to dilute Akis in combination with whatever local anaesthetic normally results in milky solutions and therefore is not recommended; (ii) using bicarbonate, optimal solubility was obtained combining Akis with lidocaine, both 1 and 2%, or mepivacaine, both 1 and 2%, whereas solutions were turbid in combination with bupivacaine, levobupivacaine, or ropivacaine. Thus, we recommend that Akis is used in combination with lidocaine or mepivacaine in a bicarbonate vehicle.

220 THE ESTROGEN-LIKE EFFECT OF 2-METHOXYESTRADIOL (2-ME) IN IN VITRO AND IN VIVO MODELS

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-oestradiol, interacts with oestrogen receptors and microtubules and has a low affinity for oestrogen receptors (ER). It has attracted considerable interest due to its potential anti-cancer therapeutic effects. 2-ME is also recognised for its unique and profound actions on various tumour cell lines and cancer independent of the hormone receptor status. Regardless of differences in function, 2-ME has an affinity for ER, however, the exact mechanisms of 2-ME action via the ER are not fully understood. In the current study, we examined the estrogenic effect of 2-ME on mRNA levels of CaBP-9k, ER, and progesterone receptor (PR) in the absence or presence of the 17β-oestradiol (E2) and progesterone (P4) in both in vivo and in vitro models by real-time RT–PCR. In vitro, cells (n = 3 per group) were exposed to a single dose of E2 (10–9 M), P4 (10–6 M), 2-ME (10–8 M, 10–7 M, 10–6 M). The mechanism of CaBP-9k induction by these chemicals pre-treated with 10–7 M ICI 182, 780 and 10–6 M RU 486 for 30 min before exposure to E2 and 2-ME were analysed. In vivo, 35 female ICR mice (PND 14 days) were divided into 7 groups (n = 5 per group), and each group was administered subcutaneously with 24% DMSO, 38% ethanol, and 38% sterile saline as a vehicle, E2 [40 μg kg–1 of body weight (BW)] a physiological dose level), 2-ME (4, 40, and 80 mg kg–1 of BW) for 3 days. The mice were killed 24 h after the final injection. To investigate the effect of antagonism, 10 mice were injected SC with ICI 182 780 (10 mg kg–1 of BW) and RU 486 (10 mg kg–1 of BW) at 30 min before injection with 2-ME (40 mg kg–1 of BW) for 3 days and killed 24 h after the final injection. Results are presented as mean ± s.e.m.; P-values were calculated using one-way ANOVA. In GH3 cells, the mRNA level of CaBP-9k was induced in the E2 (10–9 M) treatment group, and expression of CaBP-9k was also up-regulated in the 2-ME (10–7 M)-treated group. Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2-ME (40 mg kg–1 of BW) group, similar to the response with E2 (40 μg kg–1 of BW) in mice. As a blocker for ER and PR activity, ICI 182 780 and RU 486 reversed the E2 or 2-ME mediated increase of CaBP-9k and Ltf mRNA expression. We found that 2-ME significantly increased the levels of ERa and PR transcripts. In parallel with in vitro results, the mRNA levels of ERa and PR were induced by treatment with E2 and 2-ME. Taken together, our findings demonstrated that expression of estrogenic markers, CaBP-9k and Ltf, was regulated by 2-ME in both in vitro and in vivo, which may increase their estrogenic activities in female during the cycle through ER and/or PR-mediated pathway.

Exendin-4 effects on islet volume and number in mouse pancreas

The aim of this study was to evaluate Exendin-4 (EX-4) effects on islet volume and number in the mouse pancreas. Thirty-two healthy adult male NMRI mice were randomly divided into control and experimental groups. EX-4 was injected intraperitoneally (i. p.) at doses of 0.25 (E1 group), 0.5 (E2 group), and 1 µg/kg (E3 group), twice a day for 7 consecutive days. One day after the final injection, the mice were sacrificed, and the pancreas from each animal dissected out, weighed, and fixed in 10% formalin for measurement of pancreas and islet volume, and determination of islet number by stereological assessments. There was a significant increase in the weight of pancreases in the E3 group. Islet and pancreas volumes in E1 and E2 groups were unchanged compared to the control group. The E3 group showed a significant increase in islet and pancreas volume (P < 0.05). There were no significant changes in the total number of islets in all three experimental groups. The results revealed that EX-4 increased pancreas and islet volume in non-diabetic mice. The increased total islet mass is probably caused by islet hypertrophy without the formation of additional islets.

Establishing Intra- and Inter-Rater Agreement of the Face, Legs, Activity, Cry, Consolability Scale for Evaluating Pain in Toddlers During Immunization

BACKGROUND: The Face, Legs, Activity, Cry, Consolability (FLACC) scale is a five-item tool that was developed to assess postoperative pain in young children. The tool is frequently used as an outcome measure in studies investigating acute procedural pain in young children; however, there are limited published psychometric data in this context.OBJECTIVE: To establish inter-rater and intrarater agreement of the FLACC scale in toddlers during immunization.METHODS: Participants comprised a convenience sample of toddlers recruited from an immunization drop-in service, who were part of a larger pilot randomized controlled trial. Toddlers were video- and audiotaped during immunization procedures. The first rater scored each video twice in random order over a period of three weeks (intrarater agreement), while the second rater scored each video once and was blinded to the first rater’s scores (inter-rater agreement). The FLACC scale was scored at four time-points throughout the procedure. Intraclass correlation coefficients were used to assess agreement of the FLACC scale.RESULTS: Thirty toddlers between 12 and 18 months of age were recruited, and video data were available for 29. Intrarater agreement coefficients were 0.88 at baseline, 0.97 at insertion of first needle, and 0.80 and 0.81 at 15 s and 30 s following the final injection, respectively. Inter-rater coefficients were 0.40 at baseline, 0.95 at insertion of first needle, and 0.81 and 0.78 at 15 s and 30 s following the final injection, respectively.CONCLUSIONS: The FLACC scale has sufficient agreement in assessing pain in toddlers during immunizations, especially during the most painful periods of the procedure.