Diabetes-related foot disease (DFD) is a leading cause of the Australian disease burden. The 2011 Australian DFD guidelines were outdated. We aimed to develop methodology for systematically adapting suitable international guidelines to the Australian context to become the new Australian evidence-based guidelines for DFD.We followed the Australian National Health Medical Research Council (NHMRC) guidelines for adapting guidelines. We systematically searched for all international DFD guideline records. All identified records were independently screened and assessed for eligibility. Those deemed eligible were further assessed and included if scoring at least moderate quality, suitability and currency using AGREE II and NHMRC instruments. The included international guidelines had all recommendations extracted into six sub-fields: prevention, wound classification, peripheral artery disease, infection, offloading and wound healing. Six national panels, each comprising 6–8 multidisciplinary national experts, screened all recommendations within their sub-field for acceptability and applicability in Australia using an ADAPTE form. Where panels were unsure of any acceptability and applicability items, full assessments were undertaken using a GRADE Evidence to Decision tool. Recommendations were adopted, adapted, or excluded, based on the agreement between the panel’s and international guideline’s judgements. Each panel drafted a guideline that included all their recommendations, rationale, justifications, and implementation considerations. All underwent public consultation, final revision, and approval by national peak bodies.https://www.diabetesfeetaustralia.org/new-guidelines/We screened 182 identified records, assessed 24 full text records, and after further quality, suitability, and currency assessment, one record was deemed a suitable international guideline, the International Working Group Diabetic Foot Guidelines (IWGDF guidelines). The six panels collectively assessed 100 IWGDF recommendations, with 71 being adopted, 27 adapted, and two excluded for the Australian context. We received 47 public consultation responses with > 80% (strongly) agreeing that the guidelines should be approved, and ten national peak bodies endorsed the final six guidelines. The six guidelines and this protocol can be found at: New Australian evidence-based guidelines for DFD have been developed for the first time in a decade by adapting suitable international guidelines. The methodology developed for adaptation may be useful for other foot-related conditions. These new guidelines will now serve as the national multidisciplinary best practice standards of DFD care in Australia.The online version contains supplementary material available at 10.1186/s13047-022-00533-8. Diabetes-related foot disease (DFD) is a leading cause of morbidity, mortality and healthcare cost burdens in Australia –4. DFD iAustralian DFD Strategy 2018–2022 was to ensure Australia has national DFD guidelines that continually reflect up-to-date robust evidence to guide multi-disciplinary standards of DFD care [A key recommendation of the DFD care  is out-dDFD care  and has DFD care . Thus, tThe NHMRC Guidelines for Guidelines recommends developing new guidelines either from scratch (‘de novo’), or adopting and/or adapting other suitable high-quality international guidelines if no Australian equivalent is available . With noThe methodology for developing this guideline followed eight overarching methodological steps that aligned with best practice principles for adapting suitable international source guidelines as recommended by the NHMRC Guidelines for Guidelines  and the Population(s) of interest - were those defined as at risk of, or with, DFD , 6;Intervention(s) of interest - were those interventions typically used to screen, diagnose, prevent, or treat the population(s) of interest , 6;Professions to be targeted - were those multiple medical, surgical, nursing and allied health disciplines that typically provide prevention or treatment for the population(s) of interest , 19, 20;Outcomes of interest - were those outcome measures typically used for the population(s) of interest, such as ulcer healing or amputation , 6;Health care context to be targeted - were those secondary and/or tertiary health care settings and organisations that typically provide prevention or treatment for the population(s) of interest in Australia , 19, 20.The scope of these guidelines were defined using the PIPOH (spelt out below) framework recommended by ADAPTE  and baseBased on the above defined scope we performed a systematic search for potentially suitable international source guidelines , 17. TheThe title and abstract (if available) of each unique record identified from the search strategy was independently screened by three authors  for eligibility for full-text assessment. The inclusion criteria were those records: i) with the primary aim of developing clinical guidelines to prevent or manage people with, or at risk of, DFD; ii) developed for an international multi-disciplinary health professional audience; iii) written in English ; iv) based on systematic review(s) of the available literature; and v) which incorporated a final systematic review search date within 3 years of our search date (i.e. 1 May 2017) for currency. All records screened as eligible by any of the three authors were included for full text assessment.Each guideline record identified as eligible after screening then had their full text retrieved and assessed based on the same above inclusion criteria by two authors independently  . Any disRemaining eligible full text records were then independently assessed by four authors  for their methodological quality, suitability and currency to be adopted or adapted to the Australian health context , 17. TheA customised tool from NHMRC was used to assess suitability and currency . The tooAll documents that informed the development of each eligible full text guideline record were included as part of these assessments, including any systematic reviews, methodology protocols and technical reports , 17. AnyRecommendations were categorised into six sub-fieldsAll recommendations within the above suitable international source guidelines were individually extracted and evaluated to determine if they should be adopted, adapted, or excluded in the Australian context , 17, 18.b.National expert panels were convened for each sub-fieldTwo authors  independently categorised all recommendations  from all included suitable source guidelines into one of six DFD sub-fields that the authors considered the recommendation was primarily addressing , 6. The c.Panels screened all recommendations in their sub-fieldAll recommendation(s) , including rationale, summary(s) of evidence, evidence statements, quality of evidence summaries, risk of bias tables, evidence tables), were forwarded to the relevant sub-field national expert panel to screen. Each national expert panel comprised 6–8 members and was chaired by an author with relevant (inter)national research and/or clinical practice expertise in the sub-field. Panel membership included 4–6 members with either (inter)national research and/or clinical practice sub-field expertise from different disciplines, states (or territories) and genders, plus, a consumer and an Aboriginal and Torres Strait Islander representative with expertise in DFD. An (inter)national research expert was defined as having published in peer-reviewed journals and/or been ranked as an Australian expert in the sub-field according to Expertscape . An  in their sub-field for acceptability and applicability in the Australian health context, using a customised ADAPTE evaluation form  Evidence to Decision (EtD) template . The discussion sections typically included summaries of the similarities and differences between the new Australian guideline, previous Australian guideline, and the source guideline in terms of recommendations and rationale. The final draft guideline manuscript was approved via consensus of each panel.Each draft guideline manuscript was then peer-reviewed by at least one author not involved in the sub-field panel, to identify any obvious discrepancies in the recommendations or supporting reasons for the recommendations. If the author(s) identified any discrepancies, the panel was asked to revise the manuscript to consider and address the discrepancy. The agreed final draft of the sub-field guideline manuscript was deemed the consultation draft.The six draft guideline manuscripts used for public consultation , plus this guideline development protocol manuscript, were collated and formatted for consistency ready for public consultation as the new Australian evidence-based guidelines for DFD.. Additionally, a customised public consultation survey based on example surveys from the ADAPTE framework were developed for each guideline to more efficiently gain and collate aggregated feedback from the public consultation process  Supplem. FinallyPublic consultation was targeted towards Australian health professionals assessing and managing patients with DFD as well as national peak bodies/organisations representing health professionals, consumers or Aboriginal and Torres Strait Islanders. The consultation material included the six guideline manuscripts, the guideline development protocol manuscript and the consultation surveys. Public consultation for each guideline was open for a minimum period of 4 weeks and notification of the consultation period was posted weekly on DFA and/or Australian Diabetes Society (ADS) web and social media sites, plus, invitations were sent electronically to relevant national peak bodies for health professionals, consumers or Aboriginal and Torres Strait Islander people. Any Australian health professional or national peak body with an interest was encouraged to respond.https://www.diabetesfeetaustralia.org/new-guidelines/ [At the conclusion of the public consultation period all consultation survey responses were collated and analysed. The authors disseminated all aggregated survey findings and feedback comments to the relevant sub-field guideline panels for consideration and revision of their respective manuscripts accordingly. The authors subsequently quality checked each panel’s revisions . Any subdelines/ –36, submdelines/  and Austdelines/ .These new Australian guidelines will be reviewed every 2 years by the authors, or equivalent national guidelines committee, to determine by consensus if any substantial new evidence has been published that contradicts or substantially enhances any existing recommendations. If that is deemed to be the case, or after 4 years (i.e. 2025), whichever comes first, we recommend updating these guidelines using similar adaptation methodology used to develop these new guidelines or develop new guidelines de novo if the requisite funding becomes available.To try and facilitate improved implementation into clinical practice, clinical pathways were developed for each guideline that incorporated the recommendations from that guideline. The process used for developing the clinical pathways was that advocated by Flores et al. (2019) . This prFigure Table Table Table In summary, after screening all 100 IWGDF source guideline recommendations the panels deemed 68 (68%) were acceptable and applicable to the Australian health context and were adopted without further assessment. The other 32 (32%) were judged to have unsure (or no) acceptability and/or applicability in the Australian health context and required full assessment, including nine in offloading, eight in prevention, seven in infection, four in wound healing interventions, three in wound classification, and one in PAD. After full assessment of those 32 recommendations, three more were adopted, 27 adapted and two excluded for the Australian context. Therefore, of 100 IWGDF source guideline recommendations, 71 (71%) were adopted, 27 (27%) adapted and 2 (2%) excluded in the Australian guidelines. The six individual sub-field guidelines report details of all decisions for each sub-field –36.Overall, of those 27 adapted recommendations, nine were in offloading, six in prevention, four in infection, four in wound healing interventions, three in wound classification and one in PAD. The main reasons for adapting recommendations included: 20 had wording changed to be considered acceptable in Australia; ten had quality of evidence changed; four had strength of the recommendation changed; four had wording changed to be considered feasible in Australia; and/or, one had the direction for the balance of effects changed. Of the two recommendations excluded, one was in the prevention and one the infection guideline , 34. The2021 Australian evidence-based guidelines for diabetes-related foot disease. Detailed reasoning behind all 98 recommendations included in the guidelines are described in those six sub-field guideline manuscripts. Therefore, we refer all Australian health professionals caring for people with, or at risk of, DFD, to the 2021 Australian evidence-based guidelines for diabetes-related foot disease [In total, alongside this guideline development protocol manuscript, six sub-field guideline manuscripts were drafted . Collectively these form the new  disease –36.Table 2021 Australian evidence-based guidelines for diabetes-related foot disease by ten national peak bodies including the Australian Podiatry Association, Wounds Australia, Australian and New Zealand Society for Vascular Surgery, Australasian Society for Infectious Diseases, Australian Orthotic Prosthetic Association, Pedorthic Association of Australia, Australian Advanced Practicing Podiatrists - High Risk Foot Group, Australian Aboriginal and Torres Strait Islander Diabetes-related Foot Complications Program, the Australian Diabetes Society and DFA. The final endorsed guidelines, including pathways, are displayed in full on the DFA website https://www.diabetesfeetaustralia.org/new-guidelines/ [Based on the collated public consultation feedback, the guideline manuscripts were finally revised and approved by the relevant national panel and authors. The final manuscripts were endorsed as the delines/ –36, regidelines/  and weredelines/   source guidelines to the Australian health context. Of the 100 original IWGDF recommendations, 71 were adopted, 27 adapted and two excluded for use in Australia across six guideline manuscripts. These guidelines have now been endorsed by ten national peak bodies to serve as the new national guidelines and multidisciplinary best practice standards for the provision of DFD care within Australia –36.”) [There are some marked differences between these new 2021 Australian DFD guidelines –36 and t”) . This fu”) . Whereas”) –36. This”) , 26, 27,”) , 16. Las”)  and 2019”) –44 for t”) –36.Adapting the IWGDF guidelines may also be seen as a strength for the new 2021 guidelines due to the breadth of coverage and methodological quality provided by the IWGDF source guidelines –44, plusIn future iterations of these guidelines, we would hope to either align an updated adaptation of new Australian guidelines more closely with the development of the new 2023 IWGDF source guidelines  or obtaiNew Australian DFD guidelines have been developed for the first time in 10 years using best practice methodology. Over 30 national experts systematically evaluated an existing high-quality international IWGDF DFD source guideline and made necessary adaptations to be applicable and acceptable to Australian clinical contexts. A minimum of a month-long public consultation process occurred with feedback transparently incorporated where appropriate. These new DFD guidelines are endorsed by ten national peak bodies. The authors strongly urge all Australian health professionals from all disciplines caring for people at risk of or with DFD to implement all new guideline recommendations that accompany this guideline development protocol to help reduce the large national burden of DFD in Australia.Additional file 1: Table S1: Customised tool for assessing a guidelines suitability to adopt or adapt. Table S2: Public consultation survey example. Table S3: The completed ADAPTE Checklist of adapted guideline content. Figure S1: Customised ADAPTE Evaluation of acceptability and applicability form. Figure S2: Customised GRADE Evidence to Decision template.


We carried out a qualitative and quantitative analysis of the phytochemical composition of the fruits of large cranberry cultivars ‘Ben Lear’, ‘Bergman’, ‘Kalnciema agra’, ‘Lemunyon’, ‘Pilgrim’, ‘Stevens’, and ‘Tina’ grown in Latvian climatic conditions. The following predominant compounds were found in cranberry fruit samples: peonidin-3-O-galactoside, peonidin-3-O-arabinoside, cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, myricetin-3-galactoside, quercetin-3-galactoside, quercetin-3-α-L-arabinofuranoside, quercetin 3-rhamnoside, ursolic acid, and oleanolic acid. During the berry ripening period (from 16 August until 15 September), a trend of decreasing amounts of compounds was found in the fruit samples of the studied cranberry cultivars: the total amount of proanthocyanidins decreased by 1.3 times, the total amount of the identified flavonols decreased by 1.3 times, the total amount of triterpenoids decreased by 1.2 times, and the total amount of chlorogenic acid decreased by 1.7 times. During the period from 16 August until 15 September, the total amount of anthocyanins in the cranberry fruit samples increased by 2.6 to 17 times. The highest total amount of anthocyanins (5305.80 ± 27 µg/g) was detected in fruit samples of the cranberry cultivar ‘Kalnciema agra’ collected on 15 September. The amount of biologically active compounds in cranberry fruit samples varies during berry ripening. Thus, the choice of the picking time is one of the factors that determines the phytochemical composition of raw cranberry material. Vaccinium macrocarpon Ait.) are one of the widely cultivated berry crops in the food industry and are also used for the production of medicinal preparations and food supplements [Large cranberries  detected in large cranberry fruit determine their multifaceted pharmacological effects . Peron ePhytochemical composition studies are important for preparing high-quality raw plant material of the cultivated cranberry fruit, performing cranberry cultivar selection, and breeding new cultivars with more biologically active compounds in their fruits. The composition of secondary metabolites in cranberry fruit depends on the genetic characteristics of the plant and the effects of climatic conditions such as temperature, light, the chemical composition of the soil, and moisture . RecentlThe aim of our study was to investigate the phytochemical composition of anthocyanins, flavonols, chlorogenic acid, proanthocyanidins, and triterpenes in the large cranberry cultivars ‘Ben Lear’, ‘Bergman’, ‘Kalnciema agra’, ‘Lemunyon’, ‘Pilgrim’, ‘Stevens’, and ‘Tina’ grown in Latvia as well as the qualitative and quantitative changes of these compounds during berry ripening. The obtained results are important and provide new knowledge about changes in the phytochemical composition of cranberry fruit during berry ripening. These results can be used for further research in selecting suitable cranberry cultivars for their introduction and selection. Anthocyanins and anthocyanidins are a group of biologically active compounds found in cranberry fruit samples that determine their antioxidant and anti-inflammatory effects ,18. We cThe evaluation of the qualitative and quantitative compositions of anthocyanins and anthocyanidins showed that the samples of large cranberry cultivars contained four main glycoside compounds of the anthocyanin group: peonidin-3-O-galactoside, peonidin-3-O-arabinoside, cyanidin-3-O-galactoside, and cyanidin-3-O-arabinoside. The amount of peonidin-3-O-galactoside in the cranberry fruit samples during ripening varied from 6.77% to 36.24%, the amount of peonidin-3-O-arabinoside varied from 0.89% to 11.42%, the amount of cyanidin-3-O-galactoside varied from 26.66% to 50.32%, and the amount of cyanidin-3-O-arabinoside varied from 18.18% to 32.42%. Viškelis et al. studied fruit samples of cranberries grown in Lithuanian climatic conditions and found that the quantitative composition of the main anthocyanins was 32.7% peonidin-3-galactoside, 6.7% peonidin-3-arabinoside, 20.5% cyanidin-3-galactoside, and 19% cyanidin-3-arabinoside [The evaluation showed that the total amount of anthocyanins and anthocyanidins in the cranberry fruit samples ranged from 222.79 ± 2 µg/g to 5305.80 ± 27 µg/g. The highest total amount of anthocyanins (5305.80 ± 27 µg/g) was found in fruit samples of the cranberry cultivar ‘Kalnciema agra’ collected on 15 September. The lowest total amount of anthocyanins (222.79 ± 2 µg/g) was found in fruit samples of the cranberry cultivar ‘Pilgrim’ collected on 16 August, which was not statistically significantly different from the amount detected in the fruit samples of the cranberry cultivar ‘Tina’ collected on 16 August .Our study showed that from 16 August until 15 September the total amount of anthocyanins in the fruit samples of all the studied cultivars increased, with statistically significant differences between the samples of different studied cultivars. Our research showed that from 16 August to 1 September, the increase in the amount of anthocyanins was the most intense. In fruit samples of the cranberry cultivar ‘Kalnciema agra’, the total amount of anthocyanins increased by 2.5 times, and in fruit samples of the cranberry cultivar ‘Pilgrim’ the total amount of anthocyanins increased by 12 times. During the period from 1 September to 15 September, the total amount of anthocyanins in cranberry fruit increased, on average, by about 1.5 times in the samples of all the tested cranberry cultivars.As cranberry fruit ripen, the amount of anthocyanins in cranberry fruit samples increases . At the Vvedenskaya et al. found in their study that the amount of anthocyanins in cranberry fruit samples of the ‘Ben Lear’ cultivar began to increase intensely from August 18, and in the cranberry fruit samples of the ‘Stevens’ cultivar, this increase occurred 3 weeks later on September 20 . During The fruit samples of the cranberry cultivar ‘Kalnciema Agra’ collected on 1 September differed from the samples of the other studied cultivars, as they had a higher content of malvidin, its glycosides, and delphinidin-3-O-galactoside. Fruit samples of the ‘Kalnciema agra’ cultivar contained 3 times more malvidin-3-O-galactoside, 2.2 times more malvidin-3-O-arabinoside, 3 times more malvidin, and 1.5 times more delphinidin-3-O-galactoside . The craThe amount of anthocyanins in cranberry fruit samples increased during berry ripening. Thus, the choice of the time for preparing cranberry fruit is one of the most important factors that determines the qualitative and quantitative compositions of anthocyanins in raw cranberry material. Considering the high total amounts of anthocyanins and malvidin group compounds found in fruit samples of the cranberry cultivar ‘Kalnciema agra’, this cultivar can be chosen for further introduction and the selection of cranberry cultivars.Flavonols are a group of biologically active compounds whose antioxidant, cardioprotective, antibacterial, antiviral, and anticancer activities have been confirmed in both in vitro and in vivo studies ,23,24. AThe evaluation of the quantitative composition of flavanols showed that, in fruit samples of cranberry cultivars grown in Latvia, myricetin-3-galactoside, quercetin-3-galactoside, quercetin-3-α-L-arabinofuranoside, and quercetin 3-rhamnoside accounted for 91.16–93.85% of the total amount of the identified flavonols. In fruit samples, quercetin-3-galactoside accounted for 39.63% ± 4.36%, myricetin-3-galactoside accounted for 25.03% ± 4.36, quercetin-3-α-L-arabinofuranoside accounted for 17.80 ± 3.06%, and quercetin 3-rhamnoside accounted for 9.94% ± 2.52% of the total amount of flavonols. In their study, Wang et al. found that the quercetin-3-galactoside in the fruit samples of cranberry cultivars ‘Early Black’, ‘Howes’, ‘Ben Lear’, ‘Stevens’, ‘#35’, ‘Crimson Queen’, ‘Demoranville’, and ‘Mullica Queen’ accounted for 31–46%, myricetin-3-galactoside accounted for 19–32%, quercetin-3-α-L-arabinofuranoside accounted for 7–17%, and quercetin 3-rhamnoside accounted for 7–14% of the total amount of the identified flavonols .The analysis of the quantitative composition showed that the total amount of flavonols in the fruit samples of the studied cranberry cultivars varied from 1256.45 ± 22 µg/g to 4327.29 ± 113 µg/g . The higDuring the ripening of cranberries (from 16 August until 15 September), the total amounts of the identified flavonols in the fruit samples of the studied cultivars ‘Ben Lear’, ‘Bergman’, ‘Kalnciema agra’, ‘Lemunyon’, ‘Pilgrim’, ‘Stevens’, and ‘Tina’ decreased by an average of 1.3 times . Wang etWe found that the quantitative compositions of the flavonols in the cranberry samples changed only slightly during berry ripening. The results of our study correlate with those obtained by other researchers. Studies of the quantitative compositions of flavonols during berry ripening are important in the preparation of raw cranberry fruit material. They provide an opportunity to prepare a raw material with a known amount of flavonols, which can influence the biological effects of the preparations.The compounds of the proanthocyanidin group detected in cranberry fruits determine the biological effects of the preparations. A-type procyanidins suppress the adhesion of pathogenic bacterial strains to urinary tract epithelial cells . Li et aUsing the spectrophotometric method, we found that the total amounts of proanthocyanidins in the fruit samples of the studied cranberry cultivars ranged from 3.28 ± 0.11 mg EE/g to 5.99 ± 0.21 mg EE/g a. The hiDuring the berry ripening period (from 16 August until 1 September), a trend of a 1.2-fold decrease in the amount of proanthocyanidins was found in the fruit samples of cranberry cultivars ‘Ben Lear’, ‘Bergman’, ‘Lemunyon’, ‘Pilgrim’, and ‘Stevens’. In fruit samples of the cultivars ‘Kalnciema agra’ and ‘Tina’ bred in Latvia, the concentrations of proanthocyanidins increased from 4.77 ± 0.09 mg/g to 5.06 ± 0.13 mg/g and from 4.39 ± 0.13 mg/g to 4.58 ± 0.14 mg/g, respectively a. DuringVvedenskaya et al. and Wang et al. found that the concentration of proanthocyanidins decreased at the beginning of fruit ripening and slightly increased during the later stage of the ripening ,21. In tStudies of the quantitative compositions of proanthocyanidins during berry ripening are important in the preparation of raw cranberry fruit materials. Research on the quantitative compositions of proanthocyanidins enables the preparation of high-quality raw material of cranberry fruits for the production of cranberry preparations and food supplements.Chlorogenic acid has been found in the raw material of cranberry fruits. Galvez et al. indicated that chlorogenic acid reduces blood pressure, obesity, and dyslipidemia . OszmianThe analysis of the quantitative composition of chlorogenic acid in the fruit samples of the cranberry cultivars grown in Latvia showed that the quantitative composition of chlorogenic acid varied from 105.54 ± 6 µg/g to 399.04 ± 9 µg/g b. The hiDuring the investigation period (from 16 August until 15 September), the amounts of chlorogenic acid in the fruit samples of the ‘Ben Lear’, ‘Bergman’, ‘Lemunyon’, ‘Pilgrim’, ‘Stevens’, and ‘Tina’ cultivars decreased by an average of 1.7 times b. The vaThe determination of the chlorogenic acid content in cranberry plant material can be applied for the assessment of quality and sensory properties of the berries. When preparing raw cranberry fruit material, the determination of the quantitative composition of chlorogenic acid enables a better prediction of the biological effects of the produced preparations.Triterpene compounds are a group of biologically active compounds that have been shown to have dysmetabolism-preventing, anti-inflammatory, anticancer, and antioxidant effects ,31. UsinAn analysis of the quantitative compositions of triterpene compounds was performed in the fruit samples of the large cranberry cultivars ‘Ben Lear’, ‘Bergman’, ‘Kalnciema agra’, ‘Lemunyon’, ‘Pilgrim’, ‘Stevens’, and ‘Tina’ grown in Latvia. We found that ursolic acid accounted for 68.31 ± 0.95% of the total quantitative composition of the triterpene compounds and phytosterols. In the fruit samples of the studied cranberry cultivars, the amounts of other triterpenoid compounds were significantly lower: the amount of oleanolic acid was 15.75 ± 0.95%, the amount of corosolic acid was 1.21 ± 0.37%, the amount of maslinic acid was 0.17 ± 0.13%, and the amount of α-amyrin was 0.52 ± 0.30%. The analysis of the quantitative composition of triterpenes revealed that the amounts of triterpenoids in the cranberry fruit samples ranged from 4215.78 ± 45 µg/g to 6232.74 ± 123 µg/g . The higThe contents of β-sitosterol in the fruit samples of cranberry cultivars ranged from 733.88 ± 21 µg/g to 1040.98 ± 33 µg/g. The highest β-sitosterol content (1040.98± 33 µg/g) was detected in the fruit samples of the ‘Bergman’ cultivar collected on 16 August, while the lowest β-sitosterol content (733.88 ± 21 µg/g) was found in the fruit samples of the ‘Kalnciema agra’ cultivar collected on 15 September, which was not statistically significantly different from the amount detected in the fruit samples of the cranberry cultivar ‘Ben Lear’ collected on 15 September.During the study period (from 16 August until 15 September), the contents of ursolic acid, oleanolic acid, and β-sitosterol in the cranberry fruit samples decreased by about 1.2 times. Oszmianski et al. found that during the ripening period the concentrations of triterpenoids  in cranberry fruit samples of the ‘Pilgrim’, ‘Stevens’, and ‘Ben Lear’ cultivars increased by an average of 1.2 times from the beginning of berry ripening to the fully ripe stage of the berries .The quantitative compositions of corosolic acid, maslinic acid, β-amyrin, and α-amyrin in the cranberry fruit samples of the tested cultivars varied during ripening. The highest amount of maslinic acid (31.80 ± 1 µg/g) was found in the ‘Kalnciema agra’ samples collected on 15 September, and the highest amount of corosolic acid (136.26 ± 2 µg/g) was found in ‘Kalnciema agra’ samples collected on 15 September, which was not statistically significantly different from the amount detected in the fruit samples of the ‘Bergman’ cultivar collected on 16 August. In the fruit samples of all studied cranberry cultivars, during the ripening period (from 16 August until 15 September), the amount of α-amyrin varied. In the fruit samples of the ‘Ben Lear’ cultivar, it increased to 24.42 ± 2 µg/g, in the ‘Bergman’ cultivar it increased from 19.99 ± 1.5 µg/g to 51.02 ± 0.8 µg/g, in the ‘Kalnciema agra’ cultivar it increased from 23.52 ± 2 µg/g to 34.18 ± 2 µg/g, in the ‘Lemunyon’ cultivar it increased from 25.62 ± 3 µg/g to 80.79 ± 4 µg/g, in the ‘Pilgrim’ cultivar it increased from 28.00 ± 1 µg/g to 47.67 ± 2 µg/g, in the ‘Stevens’ cultivar it increased from 24.56 ± 2 µg/g to 29.77 ± 3 µg/g, and in the fruit samples if the ‘Tina’ cultivar it increased from 23.54 ± 1.6 µg/g to 55.52 ± 3 µg/g. β-amyrin was detected in the fruit samples of the ‘Lemunyon’, ‘Bergman’, ‘Pilgrim’, and ‘Stevens’ cranberry cultivars. The highest β-amyrin content (34.03 ± 3 µg/g) was detected in the fruit samples of the cranberry cultivar ‘Lemunyon’ collected on 1 September. During the study period (from 16 August until 15 September), the amounts of this compound in the cranberry fruit samples of the ‘Lemunyon’ and ‘Bergman’ cultivars were found to be, respectively, two and four times lower. β-amyrin was not detected in the fruit samples of the ‘Pilgrim’ or ‘Stevens’ cultivars collected on 15 September .Studies of the phytochemical compositions of cranberry plant raw materials enable the identification of triterpene compounds, their quantitative compositions, and trends in their variability. Cranberries are a botanical source of ursolic acid. Thus, cranberry plant material could be used in the development and production of preparations with ursolic acid. In order to evaluate the quantitative variability in anthocyanins, flavonols, proanthocyanidins, chlorogenic acid, and triterpene compounds found in the samples of large cranberry cultivars during the ripening of cranberry fruit, a principal component analysis was performed . Two priThe fruit samples of large cranberries collected on 16 August were located in the negative II PC area b. DuringThe arrangement of the cranberry fruit samples collected on 1 September was shifted to the center of the chart of the principal component analysis b. DuringThe arrangement of fruit samples of the cranberry cultivars collected on 15 September shifted to the negative I PC and positive II PC area b. The amForney et al. found that during the ripening of cranberry fruit the changes in the total amounts of phenolic compounds were not large, but changes in the compositions of individual phenolic compounds may have a greater significance for the nutritional value of cranberries . During As cranberry fruits ripen, the composition of the biologically active compounds changes. Thus, determining the phytochemical composition of raw cranberry plant material is important in order to provide the consumer with herbal preparations of high commercial value and known compositions. The highest amounts of anthocyanins in the cranberry fruit samples were detected during the third picking, and the highest amounts of triterpene compounds, flavonols, proanthocyanidins, and chlorogenic acid were detected at the early stage of berry ripening. The selection of the time of harvesting raw material for cranberry fruit is one of the important factors that determines the compositions of biologically active compounds in the berries. By applying the principal component analysis, we found that raw cranberry material prepared at the beginning of September will have a higher content of proanthocyanidins, flavonols, and chlorogenic acid. Raw cranberry material prepared in the second half of September will have a large amount of anthocyanins. Routine studies of the qualitative and quantitative compositions of the biologically active compounds of cranberry plant material would allow for better control of the compositions of cranberry plant material and the preparations made from it and for the prediction of specific pharmacological effects ,37.v/v) , formic acid , acetonitrile , hydrochloric acid , and 4-(Dimethylamino)cinnamaldehyde . Reference standards for maslinic acid, corosolic acid, oleanolic acid, ursolic acid, β-amyrin, α-amyrin, β-sitosterol, chlorogenic acid, myricetin, quercetin-3-rhamnoside, quercetin-3-α-L-arabinofuranoside, and quercetin-3-α-L-arabinopyranoside were obtained from Sigma-Aldrich . Reference standards for delphinidin-3-galactoside, cyaniding-3-galactoside, cyaniding-3-glucoside, cyaniding-3-arabinoside, peonidin-3-galactoside, peonidin-3-arabinoside, peonidin-3-glucoside, malvidin-3-galactoside, malvidin-3-arabinoside, cyanidin chloride, peonidin chloride, malvidin chloride, and myricetin-3-galactoside were purchased from Extrasynthese . Reference standards for quercetin-3-galactoside and quercetin were obtained from Carl Roth . The reference standard for quercetin-3-O-glucoside was obtained from Biochemistry . The purified deionized water used in the experiments was prepared using a Milli-Q®  water purification system.The following reagents were used in the study: acetone, methanol , ethanol 96% (Vaccinium macrocarpon Aiton.) samples of different cultivars grown in Latvian climatic conditions, which were obtained from the collection of the Latvia University of Life Sciences and Technologies. Latvia has a midlatitude climate, transitioning from maritime to continental [The object of the study was large cranberry  with a photodiode array detector. An ACE C18 reversed-phase column  was used for the separation of the compounds. The analysis of the qualitative and quantitative compositions of the anthocyanins in the cranberry fruit was performed using the ultra-high-performance liquid chromatography methodology validated by Vilkickyte et al. . GradienThe determination of the phenolic compounds in the cranberry fruit extracts was performed according to the methodology developed and validated by Urbstaite et al. . GradienThe analysis of the compositions of the triterpene compounds in the cranberry fruits was performed using the methodology developed and validated by Sedbare et al. . GradienA compound identification analysis was performed by comparing the UV absorption spectrum of the reference standard with the UV absorption spectrum of the large cranberry matrix peaks using the same retention time and based on literature data ,46,47,48v/v). The reference solution was a DMCA solution in acidified ethanol. After 5 min, the absorption was measured with an M550 UV/Vis spectrophotometer  at λ = 640 nm. The total content of proanthocyanidins was calculated from the (–)-epicatechin (0.0625 mg/mL–1 mg/mL) calibration curve  and is expressed as mg/g (–)-epicatechin equivalent (EE) dry weight.The total content of proanthocyanidins was determined by the DMCA (4-(Dimethylamino)cinnamaldehyde) assay . First, p < 0.05 were statistically significant.Data analysis was performed using the computer software programs Microsoft Excel 2016  and SPSS Statistics 21 . During the study, the means and standard deviations (SD) of the three independent evaluations were calculated. To evaluate the differences in the amounts of anthocyanins, anthocyanidins, flavonols, chlorogenic acid, proanthocyanidins, and triterpenic compounds between samples of cranberry cultivars, a one-way analysis of variance with Tukey’s test for multiple comparisons was used. The quantitative analysis data were further analyzed by applying a principal component analysis (PCA). Differences at The choice of the time of the collection of cranberry fruit samples is one of the important factors that determines the phytochemical composition of raw cranberry material. By applying the principal component analysis, we found that raw cranberry material prepared at the beginning of September will have higher contents of proanthocyanidins, flavonols, chlorogenic acid, triterpenoids, and β-sitosterol. Raw cranberry material prepared in the second half of September will have large amounts of anthocyanins and anthocyanidins. Studies of the qualitative and quantitative compositions of the biologically active compounds of cranberry fruit samples provide an opportunity to prepare raw material with a known amounts of biologically active compounds that may influence the biological effects of the preparations.


Intrahepatic cholestasis of pregnancy (ICP) is the most common, reversible, and closely related to pregnancy condition characterized by elevated levels of bile acids (BAs) in blood serum and an increased risk of adverse perinatal outcomes. Due to the complex interactions between the mother and the fetus in metabolism and transplacental BAs transport, ICP is classified as a fetal-maternal disease. The disease is usually mild in pregnant women, but it can be fatal to the fetus, leading to numerous complications, including intrauterine death. The pathophysiology of the disease is based on inflammatory mechanisms caused by elevated BA levels. Although ICP cannot be completely prevented, its early diagnosis and prompt management significantly reduce the risk of fetal complications, the most serious of which is unexpected intrauterine death. It is worth emphasizing that all diagnostics and management of ICP during pregnancy are based on BA levels. Therefore, it is important to standardize the criteria for diagnosis, as well as recommendations for management depending on the level of BAs, which undoubtedly determines the impact on the fetus. The purpose of this review is to present the potential and importance of BAs in the detection and rules of medical procedure in ICP. Intrahepatic cholestasis of pregnancy (ICP) is the most common liver disease that develops during pregnancy ,2,3,4. IICP is diagnosed on the basis of typical clinical symptoms, laboratory abnormalities, and differential diagnosis excluding other causes of skin pruritus and liver dysfunction in the pregnant woman. The disease is not associated with abnormalities detected in imaging owing to the fact that biliary ducts are not dilated and hepatic parenchyma appears normal. Its dominant symptoms are skin pruritus and increased levels of bile acids (BAs) in the blood serum of the pregnant woman . An incrCurrently, researchers believe that the diagnosis of the disease should be made on the basis of abnormalities in liver function tests (LFTs), with or without itching, in the pregnant woman ,9,10. AlLactobacillus rhamnosus LRX01 may improve intestinal immunity in ICP offspring by inhibiting the expression of farnesoid X receptor (FXR) in the ileum.ICP is a disorder that is potentially harmful to the fetus. A clear relationship between elevated BA levels in maternal serum and fetal disorders has been confirmed in clinical practice, but the underlying mechanisms remain uncertain. One of these mechanisms is undoubtedly the inflammatory mechanism that underlies the pathophysiology of ICP ,12,13,14ICP is a disease that is characteristic of the second half of pregnancy, especially the late second and entire third trimester of pregnancy. Despite this, cases of ICP already developing in the first trimester of pregnancy have been described in the literature ,20,21,22The disease occurs at different rates; the average incidence of ICP remains at the level of 0.2–15.6%, but shows large ethnic, geographic, and seasonal variation . The higThe etiology of ICP is complex and not fully explained. It is believed that the development of ICP is the result of many factors, such as: genetic predisposition, hormonal factors, environmental factors and nutritional deficiencies, and the influence of chronic diseases on the predisposition to developing cholestasis.Seasonal incidence was noted in several works; it is more common in the winter months ,26,27,28The disease is usually mild in pregnant women, but it can be fatal to the fetus, leading to numerous complications, including intrauterine death . The maiFetal death in ICP may be caused by sudden vasoconstriction of the placental surface vessels or the development of arrhythmias induced by elevated BA levels, although pathophysiology has not been confirmed ,39,40.The adult human body is characterized by the presence of numerous endogenous substances, including bile pigments , Bas, and countless xenobiotic substances. All these substances are transformed and eliminated mainly through the hepatobiliary system .The total pool of BAs is the sum of the concentration of all BAs produced in the human body, including cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), lithocholic acid (LCA), and hyodeoxycholic acid (HDCA). Primary BAs, CA, and CDCA are the most concentrated in the blood serum, while the remaining secondary and tertiary BAs are found in very small amounts in both healthy subjects and women with ICP.Disturbances in BA homeostasis may arise from genetic mutations responsible for the transport and synthesis of these molecules, impairing hepatocyte function or intestinal changes that may be associated with intrahepatic cholestasis, gallbladder disease, glucose intolerance, and non-alcoholic and alcoholic fatty liver disease .Fasting concentration of BAs is a sensitive marker of liver disease ,47. PostBAs are synthesized in the liver and constitute the major end product of cholesterol catabolism which proceeds by a multi-enzymatic pathway involving at least 17 different enzymes. In an adult human, approximately 500 mg of cholesterol is converted into BAs per day ,52. The In addition to the two main components, bile also contains numerous conjugated organic ions that are excreted from hepatocytes via the MRP2 transporter, a product of the ABCC2 gene ,58. ThesBile produced in the liver is accumulated in the gallbladder until it is released into the gastrointestinal tract under the influence of postprandial cholecystokinin release—a peptide hormone that initiates postprandial contraction of the gallbladder . In the Enterohepatic circulation is extremely efficient; the liver takes up about 95% of BAs and the remaining 5% is excreted in the feces. This loss is replaced by de novo synthesis of BAs in the liver ,66.The basic function of BAs in the human body is above all participation in digestive processes, and additionally participation in other physiological processes occurring in the human body . These pconstitute the main important mechanism for the elimination of excess cholesterol through their synthesis and subsequent fecal excretion.BAs and phospholipids prevent cholesterol from precipitating in the gallbladder by dissolving cholesterol in the bile.they act as emulsifiers, increasing the availability of fats for pancreatic lipases, facilitating the digestion of triacylglycerols in the diet.they enable the intestinal absorption of fat-soluble vitamins ,67,69.Originally, four major functions of BAs were identified:Nevertheless, in recent years, new insights into the biological activity of BAs have been confirmed. The latest discoveries have confirmed that BAs are involved in regulating their own metabolisms, transport through enterohepatic circulation, in glucose, lipid and overall energy expenditure metabolism, and in controlling signaling events in liver regeneration ,72.After the characterization and isolation of FXR, for which bile acids are physiological ligands, the functions of Bas in the regulation of glucose and lipid homeostasis were confirmed. As indicated above, BAs binding to FXR impair the expression of genes that participate in overall BA homeostasis . However, genes that participate in BA metabolism are not the only ones that are controlled by FXR action as an effect of binding BA ,74,75.FXR controls genes that metabolize in the liver lipids , lipoproteins , glucose , and that are involved in hepatoprotection .Furthermore, except for their roles in activating FXR and lipid emulsification in the intestine, the BAs assist in various signal transduction processes through the mitogen-activated protein kinase (MAPK) pathways as well as activation of the c-JUN N-terminal kinase (JNK). The pregnane X receptor (PXR), the vitamin D receptor (VDR), and the constitutive androstane receptor (CAR) are other members of the BAs-activated nuclear receptor family. An additional receptor activated in response to BAs is the G-protein coupled BA receptor 1, GPBAR1, which might be involved in the control of obesity. GPBAR1 activation in brown adipose tissue triggers the uncoupling of protein 1, thermogenin (UCP1), which leads to increased energy expenditure ,74,75,76During intrauterine life, due to the anatomical and functional immaturity of the developing fetus’ liver, there are some functional and structural differences in the transformation and elimination of harmful substances. The main organ responsible for metabolism and elimination of metabolic products during fetal intrauterine life is the placenta ,78,79,80In utero, hepatic biosynthesis of BAs and bilirubin begins relatively early, and BAs themselves reach relatively high concentrations ,82. The The process of BA synthesis in the fetus develops before the fetus has developed mechanisms that enable the effective secretion of BAs into bile. As a result, most of the synthesized BAs pass into the fetal blood serum, then a small fraction is excreted via the fetal kidneys into the amniotic fluid, and the remainder, the greater part, is eliminated through the placenta into the mother’s body. Most of the fetal pool of BAs is eliminated from the mother’s body via the gastrointestinal tract ,85. The In physiological pregnancy, the transplacental flow of BAs is supported by a gradient of BAs and bicarbonate concentrations and proceeds from the fetus to the mother ,87. ThisIn physiologically running pregnancy, a slight increase in the total concentration of BAs in the blood serum is observed with the advancement of pregnancy. In studies that assessed the level of individual BAs, it was found that the concentration of secondary BAs did not change significantly, while the level of CDCA doubled around the time of delivery . The datThe literature also describes the phenomenon of asymptomatic hypercholanemia of pregnancy (AHP), which is defined as an increased concentration of BAs in the serum of a pregnant woman without clinical symptoms and no other laboratory abnormalities . AHP mayThe most important role in the pathomechanism of ICP is played by the increased concentration of BAs and their detergent properties, which accumulate in hepatocytes, causing damage to cell membranes and the release of aminotransferases, bilirubin, γ-glutamyl transpeptidases (GGTP), and alkaline phosphatase into the blood serum.Nevertheless, it has been shown that elevated, >10 µmol/L, postprandial BA values are observed in up to 40% of asymptomatic pregnancies, which supports the theory that pregnancy is a cholestatic state (hypercholanemia) . The achMost guidelines agree that typical clinical symptoms include pruritus of the skin, which is frequently generalized, but commonly begins and predominates on the palms and soles. The itching sensation strengthens at night, oftentimes involves the right upper quadrant pain and may be accompanied by nausea, poor appetite, sleep deprivation, or steatorrhea. Abnormalities in the biochemical functions of the liver should be identified in the absence of diseases with similar clinical symptoms and laboratory abnormalities . HoweverPregnant skin pruritus is accompanied by elevated levels of hepatic function tests or BAs, not caused by other diseases, which normalize after delivery. In addition, the Royal College of Obstetricians and Gynaecologists (RCOG) guidelines emphasize that in the presence of typical clinical symptoms and abnormal levels of hepatic function tests, an increased concentration of BAs is not necessary for the diagnosis of ICP, and the definitive confirmation of the diagnosis is the resolution of clinical symptoms and normalization of laboratory markers of liver function after delivery . It is iIn a pregnancy with ICP, the BAs flow is reversed from mother to fetus, leading to an increase in the fetal pool of BAs. The above observation may be confirmed by the results of the study conducted by Laatikainen, in which the author found a higher concentration of BAs in the umbilical cord blood of fetuses from ICP pregnancies compared to those from uncomplicated pregnancies (5.6 vs. 1.8 µg/mL) . Macias The accumulation of BAs in the maternal and fetal liver observed in ICP may lead to oxidative stress and hepatocyte apoptosis, significantly affecting the course of pregnancy ,107,108.An altered BA profile is observed in women with ICP. CA remains the major BA and its concentration is significantly higher than that of CDCA, resulting in an increase in the CA/CDCA ratio and a decrease in the percentage of CDCA in the total pool of BAs. There is also an increase in the level of secondary BAs, mainly DCA, less than that of the primary BAs, which may indicate an impairment of their enterohepatic circulation . In addiThe level of total serum BAs is currently considered to be the most sensitive and specific biochemical marker used in the diagnosis and monitoring of ICP . Some reThere are no uniform ICP diagnostic criteria among the guidelines of various scientific societies ,101,102.The main purpose of the therapy of ICP is to reduce the severity of clinical symptoms and normalize the biochemical exponents of maternal liver dysfunction and minimize the risk of complications, both fetal and neonatal.The treatment of ICP is based on various therapeutic options, which include non-pharmacological treatment, the selection of appropriate pharmacotherapy, and the choice of the method and time of delivery.Non-pharmacological treatment emphasizes the important role of bed regimen, which leads to improvement of hepatic blood flow, monitoring the well-being of the fetus by using biophysical methods , assessment of fetal movements by the pregnant woman, and monitoring of concentrations of hepatic functional tests. Many scientific societies recommend monitoring total bile acid concentrations, with specific recommendations varying from one society to another. The RCOG recommends determination of bile acid concentrations at weekly intervals . The Teaat the concentration of <40 µmol/L total bile acids, childbirth is recommended between 37 0/7 and 38 6/7 weeks of pregnancy.at the concentration of 40–99 µmol/L total bile acids, childbirth is recommended between 36 0/7 and 37 0/7 weeks of pregnancy.at the concentration of >100 µmol/L total bile acids, childbirth is recommended in the 36 0/7 week of pregnancy, or earlier in the case of other events or emergency to either the fetus, mother, or both .The decision on the timing and method of delivery in pregnancies complicated by intrahepatic cholestasis of pregnancy should take into account the advancement of pregnancy, BA levels, clinical signs and obstetric history, and should be made promptly in case of either fetal, maternal, or both, emergency:contributes to the break-up of micelles in the intestines and reduces the rate of cholesterol absorptionreduces hepatic synthesis and secretion of cholesterol,has a choleretic effect,has an anti-apoptotic effect,corrects the disturbed kinetics of transplacental bile acid transport in pregnancies complicated with intrahepatic cholestasis during pregnancy,reduces the concentration of primary bile acids in the umbilical cord blood and in the amniotic fluid.dosing is 16–21 mg/kg body weight, usually spread over 3 doses ,105,111.UDCA is the main medication in the pharmacotherapy of intrahepatic cholestasis in pregnancy. It is a naturally occurring, hydrophilic, tertiary bile acid formed in the gastrointestinal tract through the bacterial metabolism of primary bile acids. After oral use the maximum blood concentration is reached after about 30–60 min. The mechanism of action of UDCA is multidirectional:Medicaments used in the treatment of intrahepatic cholestasis in pregnancy also include: ornithine aspartate, phospholipids, dexamethasone, cholestyramine, S-adenosine-L-methionine, antihistamines, and vitamin K.ICP is the most common liver disease in pregnancy. ICP is a condition in which the dominant symptoms are skin pruritus and increased levels of BAs in the blood serum of the pregnant woman. The disease is mild in pregnant women, but it can be fatal to the fetus, leading to numerous complications, including intrauterine death. According to many clinical studies the mechanisms by which BAs affect the fetus during pregnancy are fairly well understood, but still require more in-depth attention. It is worth emphasizing that BAs are of limited importance in medical diagnostics, with the exception of ICP, on which all diagnostics as well as pregnancy management are based. Therefore, it is important to standardize the criteria for diagnosis, as well as recommendations for management depending on the level of BAs, which undoubtedly determine the impact on the fetus. Hence, scientists agree that more research is needed to translate it into therapeutic strategies. Multicenter and controlled trials with more patients are required to compare bile acid levels with fetal complications as there is increasing evidence of BAs being used as a prognostic test in women with ICP.


In addition, efficacy of HCQ treatment was retrospectively compared between high LAMP3 mRNA expression in minor salivary glands and those with LAMP3 mRNA levels comparable with healthy controls. Study results show that CQ treatment stabilized the lysosomal membrane in LAMP3-overexpressing cells via deactivation of cathepsin B, resulting in decreased apoptotic cell death. In mice with established SjD-like phenotype, HCQ treatment also significantly decreased apoptotic cell death and ameliorated salivary gland hypofunction. Retrospective analysis of SjD patients found that HCQ tended to be more effective in improving disease activity index, symptom severity and hypergammaglobulinemia in patients with high LAMP3 expression compared those with normal LAMP3 expression. Taken together, these findings suggested that by determining salivary gland LAMP3 mRNA level, a patient’s response to HCQ treatment could be predicted. This finding may provide a novel strategy for guiding the development of more personalized medicine for SjD.Hydroxychloroquine (HCQ) is a lysosomotropic agent that is commonly used for treating Sjögren’s disease (SjD). However, its efficacy is controversial because of the divergent response to the drug among patients. In a subgroup of SjD patients, lysosome-associated membrane protein 3 (LAMP3) is elevated in expression in the salivary glands and promotes lysosomal dysregulation and lysosome-dependent apoptotic cell death. In this study, chloroquine (CQ) and its derivative HCQ were tested for their ability to prevent LAMP3-induced apoptosis,  Sjögren’s disease (SjD) is a chronic exocrinopathy that leads to salivary and lacrimal gland hypofunction with resultant dry mouth and eye symptoms. SjD is considered to be a systemic autoimmune disease because of the presence of lymphocytic infiltration into affected tissues, serum autoantibodies, and hypergammaglobulinemia . IncreasChloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are lysosomotropic agents, which deactivate lysosomal function by alkalinizing the lysosomal pH. Specifically, they inhibit endolysosomal, autophagic, and endolysosomal toll-like receptor (TLR) pathways , 8. HCQ LAMP3 mRNA, encoding lysosome-associated membrane protein 3 (LAMP3), and this expression was associated with some SjD-related clinical findings, such as lymphocytic infiltration, serum autoantibodies and hypergammaglobulinemia [Transcriptome analysis of minor salivary glands of SjD patients identified increased expression of ulinemia . Unlike ulinemia . Confocaulinemia , 3. Inteulinemia . The undulinemia –4.LAMP3 expression for HCQ therapy will improve overall clinical outcome of this drug by a retrospective cohort study in combination with in vitro and in vivo experiments.As patients with SjD present with heterogeneous symptoms, recent studies have focused on stratifying them into homogeneous subsets to develop a personalized treatment strategy , 17. ConFig 1A) [LAMP3 destabilizes the lysosomal membrane via degradation of LAMP1, which leads to lysosomal membrane permeabilization (LMP), and leakage of activated lysosomal enzymes into the cytoplasm. This in turn activates caspases, resulting in apoptosis. LAMP3-associated LAMP1 degradation and LMP are dependent on cathepsin B activity (CTSB (encoding cathepsin B) , it did decrease the amount of the double heavy chain form of cathepsin B , confirming that CQ, an alkalinizing agent, can inhibit the pH-dependent maturation process of cathepsin B in the lysosome [To investigate whether CQ can inhibit LAMP3-induced apoptotic cell death, we first treated stably LAMP3-overexpressing A253 cells with CQ for 24 hours and then analyzed cathepsin B expression, LAMP1 expression, galectin-3 puncta formation (a marker of LMP), and apoptotic cell death. Pro-cathepsin B, which is synthesized on the rough endoplasmic reticulum, matures into single and double heavy chains in the late endosome and lysosome, respectively . Althouglysosome .Fig 2A), reduced galectin-3 puncta formation , and decreased apoptosis  in LAMP3-overexpressing cells compared to vehicle-treated LAMP3-overexpressing cells. These results demonstrated that CQ decreases LAMP3-induced apoptotic cell death in vitro by deactivating cathepsin B.In addition, CQ treatment restored LAMP1 expression , as previously reported [in vitro results, HCQ treatment restored LAMP1 expression . Treatment with HCQ resulted in a significant increase in salivary flow rate compared to placebo , while serum anti-Ro/SSA and anti-La/SSB antibody levels were not affected . In short, these results suggested that HCQ treatment decreased LAMP3-induced apoptotic cell death and restores salivary gland function in a mouse model of SjD.Histological analysis of the glands suggested a trend towards decreased lymphocytic infiltration in the HCQ-treated mice . For comparison, a group of 10 age-matched healthy volunteers were recruited from the same hospital.Our LAMP3 level  and 9 showed a normal LAMP3 level, comparable with that of the healthy volunteers . At baseline (before HCQ treatment), there were no significant differences in patient characteristics between the high-LAMP3 and normal-LAMP3 groups. But, the proportion of patients with a high lymphocytic focus score (≥ 3) was higher in the high-LAMP3 group , while the proportion of patients with a decreased salivary flow rate (≤ 0.1 mL/min) was higher in the normal-LAMP3 group . Most of the patients were concomitantly treated with low-dose corticosteroids  and/or methotrexate (Table 1).RNA-sequencing data showed that 5 SjD patients had a high LAMP3 group and 4 years in the normal-LAMP3 group. The European League against Rheumatism Sjögren’s syndrome disease activity index (ESSDAI) was significantly decreased after HCQ treatment in both the high-LAMP3 group  and normal-LAMP3 group . However, of the 8 patients with moderate disease activity (ESSDAI: 5–13 [LAMP3 group and 4/6 patients (67%) in the normal-LAMP3 group showed minimal clinically important improvement  in the normal-LAMP3 group . After HCQ treatment, 4/5 patients (80%) in the high-LAMP3 group and 5/9 patients (55%) in the normal-LAMP3 group reported satisfactory control of SjD symptoms such as dryness, pain, and fatigue  < 5 [Fig 5D). Analysis of clinical laboratory data showed that HCQ treatment significantly decreased serum gamma globulin levels in the high-LAMP3 group  but not in the normal-LAMP3 group . Hypergammaglobulinemia  was improved in 3/3 patients (100%) in the high-LAMP3 group and 2/5 (40%) in the normal-LAMP3 group after HCQ treatment . Severe adverse events related to HCQ use were not reported during the observation period.The median duration of HCQ treatment was 2 years in the high-AI: 5–13 ) at baseSDAI ≥ 3 ). FurthePRI) < 5 ) . All subjects provided written informed consent prior to the initiation of any study procedure.11 particles/mouse in 100 μL) of female, 2-month-old C57BL/6 mice  by retrograde ductal instillation through a thin cannula. After 4 months, mice were randomly assigned to weekly intraperitoneal HCQ injections  in a vehicle of phosphate buffered saline (PBS) at 60 mg/kg or placebo (PBS only) for 3 months, based on a previous report [LAMP3-overexpressing mice were established as described previously . Brieflys report . PilocarAt the end of the study, mice were sacrificed and blood and whole submandibular glands were collected. Serum was separated by centrifugation and then stored at -80°C. Presence of serum anti-Ro/SSA and anti-La/SSB antibodies were tested using the Mouse Anti-SSA/Ro60 Ig’s  ELISA Kit (#5710) and Mouse Anti-SSB Ig’s  ELISA Kit (#5810), respectively, according to the manufacturer’s instructions . Lymphocytic infiltration in both the submandibular glands were investigated using hematoxylin & eosin staining. The outcomes were evaluated by unblinded researchers.All procedures involving live animals were approved based on institutional guidelines and standard operating procedures following the NIH Guide for the Care and Use of Laboratory Animals .2.A253 cells —a human salivary gland cell line—were cultured in McCoy’s 5A Medium  supplemented with 10% fetal bovine serum. Stably LAMP3-overexpressing and control A253 cells were established as described previously [CTSB (Hs00947439_m1) and ACTB (Hs01060665_g1) were used (Thermo Fisher Scientific). Gene expression relative to ACTB was calculated using the ΔΔCt method.Total RNA was extracted from A253 cells using the RNeasy Mini Kit , treated with RNase-Free DNase Set (QIAGEN), and reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). To calculate transcript expression levels, TaqMan gene expression assays for PCR cycles were performed using the Quantstudio3 Real-Time PCR System  with the following conditions: 2 minutes at 50°C, 10 minutes at 95°C, 50 cycles of 15 seconds at 95°C, and 1 minute at 60°C.A253 cells were lysed in RIPA Lysis and Extraction Buffer with protease and phosphatase inhibitors  and cleared by centrifugation at 17,000 g at 4°C for 25 minutes. Supernatants were heated at 97°C in NuPAGE LDS Sample Buffer for 10 minutes, resolved by SDS-PAGE, and electrophoretically transferred to polyvinylidene difluoride membranes . Membranes were blocked with 2% non-fat dried milk at 25°C for 1 hour, and then incubated at 4°C overnight with one of the following primary antibodies: anti-cathepsin B , anti-LAMP1  or anti-α-tubulin . After washing three times, membranes were incubated with rabbit or mouse IgG horseradish peroxidase-linked whole antibody (Sigma-Aldrich) at 25°C for 1 hour. Signals were visualized using Super Signal West Pico Chemiluminescent Substrate or Super Signal West Pico PLUS Chemiluminescent Substrate (both from Thermo Fisher Scientific).A253 cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized using 0.1% Triton-X-100 (Sigma-Aldrich) for 10 minutes and blocked with 2% bovine serum albumin (BSA) for 30 minutes, all at room temperature. Then, cells were incubated with a mixture of 10 μg/mL mouse anti-galectin-3 antibody  in 2% BSA at 4°C overnight. The next day, cells were incubated with a mixture of 10 μg/mL Alexa Fluor-647 anti-mouse IgG  in 2% BSA at room temperature for 1 hour.Formalin-fixed paraffin embedded sections of murine submandibular glands were deparaffinized, rehydrated and subjected to citric acid microwave antigen retrieval. Slides were blocked with 2% BSA (Sigma-Aldrich) and permeabilized by 0.1% Triton-X-100 (Sigma-Aldrich) for 30 minutes at 25°C. Slides were incubated with mouse anti-galectin-3  and rabbit anti-LAMP1  antibodies at 4°C overnight, followed by incubation with Alexa Fluor-647 anti-mouse IgG and Alexa Fluor-488 anti-rabbit IgG  at room temperature for 1 hour.Sections were subsequently counterstained with DAPI . Images were acquired using a fluorescent microscope  and analyzed using ImageJ software .in vitro, A253 cells were stained using the APC Annexin V Apoptosis Detection Kit with 7-AAD , and apoptotic cells were detected using the BD Accuri Flow Cytometer .To detect apoptosis in vivo was visualized using a TUNEL assay  on formalin-fixed, paraffin-embedded murine submandibular gland sections, according to the manufacturer’s instructions. After counterstaining with DAPI mounting medium (Abcam), images were acquired with a fluorescent microscope (Nikon) and analyzed using ImageJ software.Apoptosis t-test. When appropriate, Dunnett’s correction was used to correct for multiple hypothesis testing. Categorical variables were compared using the Chi-squared test. P values < 0.05 were considered statistically significant. All analyses were performed using GraphPad Prism 8.0 software.Data are presented as mean ± standard deviation (SD). Quantitative variables were compared using a two-tailed paired or unpaired Student’s S1 File(PDF)Click here for additional data file.


Comfort is a primary patient objective and central to patient experience, and thus, maximising comfort is a universal goal for healthcare. However, comfort is a complex concept that is difficult to operationalise and evaluate, resulting in a lack of scientific and standardised comfort care practices. The Comfort Theory developed by Kolcaba has been the most widely known for its systematisation and projection and most of the global publications regarding comfort care were based on this theory. To develop international guidance on theory-informed comfort care, a better understanding about the evidence on the effects of interventions guided by the Comfort Theory is needed.To map and present the available evidence on the effects of interventions underpinned by Kolcaba’s Comfort theory in healthcare settings.The mapping review will follow Campbell Evidence and Gap Maps guideline and Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews Protocols guidelines. An intervention-outcome framework has been developed based on Comfort Theory and the classification of pharmacological and non-pharmacological interventions via consultation with stakeholders. Eleven electronic databases  and grey literature sources  will be searched for primary studies and systematic reviews between 1991 and 2023 written in English and Chinese as the papers regarding Comfort Theory were first published in 1991. Additional studies will be identified by reference list review of included studies. Key authors will be contacted for unpublished or ongoing studies. Two independent reviewers will screen and extract data using piloted forms with discrepancies resolved by discussion with a third reviewer. A matrix map with filters of study characteristics will be generated and presented through software of EPPI-Mapper and NVivo.More informed use of theory can strengthen improvement programmes and facilitate the evaluation of their effectiveness. Findings from the evidence and gap map will present the existing evidence base for researchers, practitioners and policy-makers and inform further research as well as clinical practices aiming at patients’ comfort enhancement.The online version contains supplementary material available at 10.1186/s13643-023-02202-8. Comfort is a primary patient objective and central to patient experience , 2, and Comfort, however, is a complex concept that is difficult to define, operationalise and evaluate. Comfort experience is transient and multidimensional and corporates more than the absence of pain . It is aComfort has not been fully conceptualised and operationalised until American researcher Dr. Katherine Kolcaba developed the Comfort Theory , 14, 15.Healthcare professionals (HCPs) assess comfort needs of patients/family members that are not met.HCPs design interventions to address those needs.Intervening variables are taken into account in designing interventions.HCPs measure comfort before and after interventionsIf comfort is enhanced, patients/families engage more fully in health-seeking behaviours (HSBs).When HSBs are enhanced, the institution integrity is enhanced.The conceptual framework of Comfort Theory  is depicIntervening variables are those factors that HCPs cannot easily change, such as extent of social support and financial resources . HSBs inApplying the Comfort Theory in care practices is called Comfort care which is a philosophy of healthcare that focuses on addressing comfort needs, a pattern for holistic care but is individualised for each recipient or group . ComfortKolcaba defines comfort as a holistic experience: “the immediate experience of being strengthened through having the needs for relief, ease, or transcendence met in four contexts: physical, psychospiritual, environmental and sociocultural contexts” ( P14). ReHolistic interventions which are meant to bring about desirable whole-person effects, such as music therapy, guided imagery , 20, masKolcaba has also developed General Comfort Questionnaire (GCQ) to measure comfort of people in general condition of illness , which rComfort is a universal concept understood across most disciplines and cultures and Comfort Theory can be adopted to any healthcare settings or age group  as visioThis evidence and gap map (EGM) will collate the existing evidence and display clusters of evidence and gaps in evidence base that will serve as a resource to guide prioritisation of further research and increase the accessibility and use of evidence for informed decision-making by stakeholders including researchers, decision-makers and practitioners.To our knowledge, there is no published EGM about the effectiveness of comfort interventions utilising Comfort Theory by Kolcaba. One scoping review investigated the use of non-pharmacological interventions (NPIs) for comforting patients in palliative care, including 10 NPIs on cancer patients: aromatherapy, reiki and therapeutic touch; aromatherapy, footsoak and reflexology; aromatherapy; aromatherapy massage; massage therapy; noncontact therapeutic touch; music therapy; hypnotherapy; art therapy; and electromyography biofeedback-assisted relaxation, with three studies evaluated the total comfort and the remaining evaluated comfort related variables . AnotherDevelop a framework of types of interventions and outcomes related to the effectiveness of these comfort interventions and practices.Describe the characteristics of included studies which summarise the intervention, population, country, setting and study design.Identify major gaps in existing evidence base.The proposed EGM will map and present existing evidence on the effects of comfort interventions and practices among adults underpinned by Comfort Theory of Kolcaba in an international scope. The specific objectives of the map are to:The Campbell Evidence and Gap Maps guideline (EGMs) will be used to systematically search and map impact evaluation and systematic reviews on the effects of interventions and practices using Kolcaba’ s Comfort Theory to promote adults’ comfort . DifferiConsistent with the standard EGM matrix, the current EGM will consist of two primary dimensions: rows listing intervention categories, and columns listing outcome types, and each cell of the matrix will show studies containing evidence on that particular combination of intervention and outcome. The map will also contain relevant filters, such as types of theory application, study design, population, region and country, focusing on a subset of studies meeting certain criteria. The proposed intervention-outcome framework, as described in the following, has been developed by the authors through a consultative process with stakeholders.Three international experts who were the founder and advocator of the Comfort Theory and practitioners in comfort care practices from the USA and China were consulted as the advisory panel. Katharine Kolcaba is an American nursing theorist and nursing professor. Dr Kolcaba is the founder of the Comfort Theory under investigation. She suggested a scope on the effectiveness of theory application in comfort enhancement and the criteria on descriptive articles about certain comfort interventions as well as the reliability and validity of instruments used to evaluate comfort scores. Dr April Bice is an American researcher and lecturer in paediatric nursing, and a promoter of Comfort Theory, and she offered potential sources of relevant evidence. Yongxing Shi is a Chinese associate professor and expert in community-based EoL care based in Shanghai, China, where formal palliative and hospice care services have been piloted across the city since 2017. The three experts made comments on the scope and framework of the present EGM, as well as the sources of evidence via face-to-face and email discussions.A matrix framework with dimensions of interventions and outcomes will be generated. Figure Technical comfort measures are those interventions designed to maintain homeostasis and manage pain; (2) Coaching is to relieve anxiety, provide reassurance and information, promote hope, listen and help plan realistically for recovery, integration, or death in a culturally sensitive way; (3) Comfort Food for the Soul are those comfort measures that target transcendence, making patients feel strengthened in an intangible, personalised sort of way [Kolcaba has proposed three categories of comfort interventions .This EGM review will include impact evaluations and systematic reviews. Impact evaluations use one of the following designs: (1) randomised controlled trials (RCT), (2) quasi-experimental design, (3) before versus after studies and (4) observational description of services or practices. Systematic review (SR) refers to the review of primary studies adopting systematic approach, which includes (a) search of at least three databases, (b) screening with explicit inclusion criteria and (c) coding and reporting of all relevant findings , such asThe review will include adult participants who aged 18 and older, of any gender, race and ethnicity. To be inclusive, studies or reviews which state a focus on adults without providing the age of participants or that serve both adults and non-adults will be included. Participants groups may be but will not be limited to patients, their family members and HCPs.Participants can be from any geographic location and any settings as defined by the World Health Organization regions  . The stuThe review will include any interventions applying Kolcaba’s Comfort Theory to enhance comfort. The application can be that the Comfort Theory was used as the theoretical framework underpinned the intervention. Alternatively comfort questionnaires derived from the Comfort Theory and TS were used to measure participants’ comfort level. This review will only consider papers that clearly indicate that the Comfort Theory of Kolcaba or related comfort questionnaires were used, with cited references of which Dr Katherine Kolcaba was listed as the author or one of the authors.As outlined in the conceptual framework above Figs.  and 3, tThe columns of the evidence map matrix will be the main outcome categories that were developed by the authors based on the Comfort Theory, comprising (1) comfort, (2) comfort-related variables (added by authors), 3) HSBs and (4) institutional integrity. HSBs include three subcategories: internal behaviours, external behaviours and peaceful death. The main outcome categories and their definitions are presented in Table  HSBs andA comprehensive search of eligible evidence will be conducted based on the framework of interventions and outcomes as outlined above, following the guidance by Campbell . First, Papers written in English and Chinese will be considered for inclusion as the research team is proficient in the two languages. Most papers published in the widely used international databases are written in English so that the consideration of papers in English allows the most extent of coverage on papers met the inclusion criteria. Databases mainly covering publications in Chinese will be searched to scope evidence from the context of China. Papers published between 1991 and 2023 will be included as the first publication regarding Comfort Theory by Kolcaba was in 1991 , 27.Following the search, all identified papers will be imported into the software Endnote X9 . After removing duplicates, two reviewers will initially screen the title and abstract of each paper against the inclusion criteria on adult population, application of Comfort Theory, intervention types and outcome measures, language and publication year and exclude those that are considered completely irrelevant. Following the screening of title and abstract, full text of potentially relevant papers will be retrieved and reviewed in detail in qualitative data analysis software NVivo  by two reviewers independently. Any disagreements that arise between the two reviewers at each stage of the study selection process will be solved through discussion with the third reviewer to achieve a final consensus.The process and criteria of screening and selection will be piloted by the reviewers. Results of search and process of paper selection will be documented and presented in a PRISMA flow diagram .Each included study will be coded and extracted by two reviewers independently in NVivo software using the standardised coding form which has been piloted and refined .Additional file 2. Coding form.