**Tumor Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS) with a New Pathogenic Variant in** *TNFRSF1A* **Gene in a Family of the Adult Male with Renal AA Amyloidosis—Diagnostic and Therapeutic Challenge for Clinicians**

**Jolanta Zegarska 1,†, Ewa Wiesik-Szewczyk 2,†, Ewa Hryniewiecka 1, Beata Wolska-Kusnierz 3, Dariusz Soldacki 2,4, Magdalena Kacprzak 5, Agnieszka Sobczynska-Tomaszewska 5, Kamila Czerska 5, Pawel Siedlecki 6,7, Karina Jahnz-Rozyk 2, Ewa Bernatowska 3, Radoslaw Zagozdzon 1,4,6,\* and Leszek Paczek 1,6,\***

**Citation:** Zegarska, J.; Wiesik-Szewczyk, E.; Hryniewiecka, E.; Wolska-Kusnierz, B.; Soldacki, D.; Kacprzak, M.; Sobczynska-Tomaszewska, A.; Czerska, K.; Siedlecki, P.; Jahnz-Rozyk, K.; et al. Tumor Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS) with a New Pathogenic Variant in *TNFRSF1A* Gene in a Family of the Adult Male with Renal AA Amyloidosis—Diagnostic and Therapeutic Challenge for Clinicians. *J. Clin. Med.* **2021**, *10*, 465. https:// doi.org/10.3390/jcm10030465

Academic Editor: Eugen Feist Received: 30 December 2020 Accepted: 19 January 2021 Published: 26 January 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

1 Department of Immunology, Transplant Medicine and Internal Diseases, Medical University of Warsaw, 59 Nowogrodzka St., 02-006 Warsaw, Poland<EMAIL_ADDRESS>(J.Z<EMAIL_ADDRESS>(E.H.)

**Abstract:** Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) belongs to systemic autoinflammatory diseases (AIDs). Many of these syndromes are genetically conditioned and can be inherited. Diagnosis relies on clinical symptoms and should be confirmed by genetic testing. One of the most serious complications is AA amyloidosis. We present the diagnostic route of a 33-year-old male with AA amyloidosis and his children, leading to diagnosis of monogenic autoinflammatory syndrome, confirmed by genetic analysis. A novel variant of the in-frame insertion type in one allele of *TNFRSF1A* gene was found by whole exome sequencing and confirmed by Sanger sequencing, which allowed a diagnosis of TRAPS. Three-dimensional modeling was used to assess the structural changes introduced into TNFR1 molecule by the insertion. The analysis of the 3D model revealed that accommodation of the 4AA insert induces misalignment of three cysteine bridges (especially the C70- C96 bridge) in the extracellular domain, leading to putatively misfolded and improperly functioning TNFR1. Three of the patient's daughters inherited the same variant of the *TNFRSF1A* gene and presented TRAPS symptoms. TRAPS is a very rare disease, but in the presence of suggestive symptoms the genetic diagnostic workout should be undertaken. Early diagnosis followed by appropriate clinical management can prevent irreversible complications.

**Keywords:** new genetic variant; monogenic autoinflammatory syndrome; diagnostic delay; anakinra; damage index; genetic inheritance; personalized therapy

#### **1. Introduction**

Monogenic autoinflammatory diseases (AIDs) cover a spectrum of syndromes, which lead to chronic or recurrent inflammation caused by activation of the innate immune system, typically in the absence of high autoantibody titers [1]. The four most common monogenic AIDs are: NLRP3-associated autoinflammatory disease (NLRP3-AID), familial Mediterranean fever (FMF), mevalonate kinase deficiency (MKD), and tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS).

TRAPS is an autosomal dominant disease, caused by mutations in *TNFRSF1A* gene which encodes the protein named tumor necrosis factor receptor 1 (TNFR1), which plays a crucial role in the inflammation and apoptosis [1]. The estimated prevalence of TRAPS is one per million. Symptoms include recurrent episodes of fever, lasting about 3 weeks, abdominal, chest, and muscle pains, skin rash, typically found on the limbs, periorbital edema, and joint pain [2]. Inflammatory markers are always elevated during acute episodes. The onset of the disease may occur at any age, from infancy to late adulthood, but most patients have their first episode in childhood. In most cases, relatives are affected, and positive family history strongly supports the diagnosis. AA amyloidosis is the main longterm complication of TRAPS. Depending on the genetic variant, it is estimated that 2–24% of untreated patients with TRAPS would develop AA amyloidosis [2–5], usually in midadulthood, but the risk of this complication has significantly been decreased by modern anti-inflammatory therapies [6]. The evaluation of patient with systemic AA amyloidosis, in whom an obvious cause cannot be identified, is a challenge in clinical practice, as there are over 100 diseases associated with AA amyloidosis. Among them, strong association is reported for AIDs. The correlation is not unexpected, because autoinflammatory syndromes often cause the long-standing inflammation [7]. However, due to low awareness and heterogenous presentation, especially in sporadic cases, the diagnosis of AIDs might be overlooked. Indeed, long diagnostic delay is usually reported. Importantly, the proper diagnosis allows for avoiding the complications and progressive, irreversible organ damage. Moreover, worse prognosis and unfavorable outcomes of renal transplantation is reported in AA amyloidosis in the course of AIDs [8].

The therapies include corticosteroid therapy, non-steroidal anti-inflammatory drugs (NSAIDs) during flares or a treatment based on biological agents, depending on the requirements of the particular patient and disease severity [9,10]. Targeted anti-inflammatory treatment is the only option in preventing further systemic deposition of amyloid and recurrence of amyloidosis in transplanted organ.

The aim of the study was the identification of molecular basis and inheritance pattern of disease in an adult male with AA amyloidosis and episodes of fever suspected of AID, as well as in the members of his family.

### **2. Materials and Methods**

#### *2.1. Patients*

4 patients were included in the study—proband and all his 3 children (daughters). At the admission, the proband was 33-year-old Caucasian male, who was referred for nephrology consultation because of proteinuria, chronic renal failure with estimated glomerular filtration rate (eGFR) 69 mL/min./1.73 m2, and biopsy-proven kidney AA amyloidosis.

#### *2.2. Methods of Molecular Analysis*

Next-generation sequencing (NGS). Whole exome sequencing (WES) was performed. Patient's genomic DNA was extracted from the whole blood sample, and sequencing library was prepared according to Agilent Sure-Select Human All Exon V5 protocol. The enriched DNA libraries were sequenced by the Illumina HiSeq4000 instrument (Illumina, Inc., Sand Diego, CA, USA). All procedures for exome sequencing were conducted by Macrogen (Seoul, Korea). Raw sequencing reads were mapped to the reference genome using BWA [11]. Duplicates were removed using Picard software (Broad Institute, Cambridge, MA, USA), and variants were named using Samtools software (SourceForge, San

Diego, CA, USA). Variants in 289 genes connected with autoinflammatory diseases were analyzed (the list of genes [12] is presented in Supplementary Table S1). The following in silico prediction software programs were used to assist with interpretation of pathogenicity of detected variant: Alamut visual v 2.9.0 (Interactive biosoftware, SOPHiAGENETICS, CH-1025 Saint Sulpice, Switzerland). The presence of the variant in control populations was checked in 1000Genomes [13], the Exome Variant Server [14], and the Exome Aggregation Consortium (Broad Institute) and gnomAD (Broad Institute).

#### *2.3. 3D Protein Modeling*

The newly identified AHRH insertion was manually introduced into the structure of TNFR1 (Tumor necrosis factor receptor superfamily member 1A; PDB:1FT4) with the MAV (Multalign Viewer) module of UCSF (University of California San Francisco, CA, USA) Chimera software (v 1.14) [15]. Next, the Modeler software [16] was used to generate 5 different models of the modified structure. The resulting models were validated with zDOPE (Discrete Optimized Protein Energy score [17]). Final model was subjected to a short minimization procedure (MMTK—Molecular Modelling Toolkit, Center for Molecular Biophysics, CNRS-Orleans, France) with Amber ff14SB force field, 100 steps of steepest descent and 10 steps conjugate gradient) to lessen the sterical constraints introduced by the modeling procedure.

### **3. Results**

*3.1. Patients*

### 3.1.1. Patient 1—Proband

33-year-old Caucasian male was referred for nephrology consultation because of proteinuria 3.2–5.7 g/day; chronic renal failure with estimated glomerular filtration rate (eGFR) 69 mL/min./1.73 m2 and biopsy-proven kidney AA amyloidosis. On admission, he complained of chronic fatigue; otherwise, he denied any other symptoms. His family medical history was reported as irrelevant. His comorbidities were as follows: arterial hypertension, hypercholesterolemia, normocytic anemia. There were no clinically relevant findings during physical examination, except for the post-appendicitis scar in right lower quadrant of the abdomen.

In routine laboratory tests, the erythrocyte sedimentation rate (ESR) was 47 mm/h (reference range: 0–12 mm/h), and serum concentration of C-reactive protein (CRP) 61.17 mg/L (reference range: up to 9 mg/L), serum amyloid A (SAA) 60.9 mg/L (reference range: up to 6.4 mg/L). Laboratory work-up for anti-nuclear (ANA), anti-neutrophil cytoplasmic (ANCA) antibodies and rheumatoid factor were negative. Blood and urine cultures were also negative.

Detailed analysis of his past medical history revealed that from the age of 7 the patient reported recurrent episodes of abdominal pain with fever up to 40 ◦C, upper respiratory tract symptoms (pharyngitis, tonsillitis), arthralgia of elbow, knee, and wrists and elevated inflammatory parameters. These episodes were recurring irregularly, at least twice a year, with fever lasting 7–10 days and abdominal pain lasting 10–14 days. He was repeatedly hospitalized and suspected of infections (at the age of 7), chronic endocarditis (aged 14), lambliasis with cholangitis (aged 13), or connective tissue diseases. Episodes of abdominal pain were the rationale for appendectomy (age 14). Inflammatory bowel disease and chronic pancreatitis were excluded. Despite extensive research, the reason for recurrent fevers was not identified. During adolescence, recurrent acute episodes stopped, but chronic low-grade fever and fatigue persisted. At the age of 28, the patient was diagnosed with proteinuria, kidney function impairment, and arterial hypertension. Kidney biopsy specimen revealed AA amyloidosis.

Based on the medical history, AIDs were included as a potential cause for AA amyloidosis. Initial suspicion of FMF was made and treatment with colchicine 0.5 mg/day twice a day was ordered, with short-time improvement of ESR and CRP. An attempt to increase colchicine dosage to 0.5 mg three times a day was unsuccessful due to diarrhea. Moreover, genetic tests for FMF did not reveal any known pathogenic variants in exons 2, 3, and 10 of *MEFV* gene. Due to unsatisfactory effectiveness of the treatment and sustained suspicion of AID as the cause of AA amyloidosis, the genetic diagnostic work-up was continued by next-generation sequencing (NGS).

#### 3.1.2. Remaining Patients

#### Patient 2

At the age of 7, Patient 1's daughter had been referred for consultation due to episodes of fever up to 39 ◦C lasting for 6–10 days accompanied by severe abdominal pain, lymphadenopathy (cervical, inguinal) and recurrent pharyngitis. Flares have been occurring irregularly 4–5 times a year since the age of 2. Her physical examination between episodes was normal. She had constantly elevated SAA during and between febrile flares (67–813 mg/dL). Until the diagnosis of the father, her symptoms were treated as typical childhood infections.

#### Patient 3

At the age of 9, the second daughter had presented fever episodes. Her symptoms have been sporadic and mild, and lasted approx. 10 days. She responded well to antipyretic treatment. Her acute inflammatory reactants were high during flares (CRP up to 6.0 mg/dL), and between episodes were within the reference range (SAA 3.7 mg/dL).

#### Patient 4

At the age of 17, the oldest daughter presented with severe abdominal pain, lowgrade fever 37.5 ◦C. No abnormalities were found on additional investigation except for abnormal inflammatory markers (CRP 8.2 mg/dL). Symptoms persisted for 3 weeks, without response on NSAID and resolved spontaneously. Otherwise, her medical history was unremarkable. Her acute inflammatory reactants between episodes were within the reference range (SAA 0.8 mg/dL, CRP 0.2 mg/dL).

#### *3.2. Genetic Results*

#### Genetic Variant Identification

As the result of WES analysis of Patient 1, the novel variant of the insertion type: c.362\_363insTGCAAGACACAG in one allele of *TNFRSF1A* gene was identified (Supplementary Figure S1). Then, the NM\_001065.5:c.362\_363insTGCAAGACACAG/p.(Arg121\_ Asp122insAlaArgHisArg) variant was confirmed by Sanger sequencing (Figure 1).

Because the NM\_001065.5:p.(Arg121\_Asp122insAlaArgHisArg) insert has been identified in the TNFR1 protein for the first time, we decided to assess the potential effects of this insertion on the protein structure by 3D modeling (Figure 2). The acquired 3D model suggested that the insert may influence three cysteine bridges, with the C70–C96 being the crucial one for correctly orienting CRD2 and CRD3 domains. Accommodation of the 4AA insert leads to misalignment cysteine bridges leading to putatively misfolded and improperly functioning TNFR1. Therefore, the NM\_001065.5:p.(Arg121\_Asp122insAlaArgHisArg) insert should be considered pathogenic. This notion warrants further investigations on the potential phenotypic changes induced by this novel TNFR1 variant under experimental conditions, preferably by comparison with some previously identified pathogenic variants, as exemplified in [18,19].

**Figure 1.** Sanger sequencing of NM\_001065.5:c.362\_363insTGCAAGACACAG variant. The alignment to wild sample sequence is visualized using Mutation Surveyor Software, v.4.0.7 (Softgenetics, State College, PA 16803, USA). The upper panel represents the reference sequence, underneath—Sanger sequencing of mutated sample—from forward (middle panel) and from reverse (bottom panel) primers.

**Figure 2.** (**A**,**B**) Comparison between the structure of wild-type variant of TNFR1 protein (**A**) and modeling of the predicted misfolding (red arrow) resulting from the NM\_001065.5: p.(Arg121\_Asp122insAlaArgHisArg) insertion in the pathogenic variant of TNFR1 (**B**).

#### *3.3. Management*

From 19 December 2017, when biologic treatment for AIDs got reimbursement in Poland, Patient 1 started treatment with an interleukin 1 inhibitor—anakinra, 100 mg subcutaneously/day, as home therapy. At the beginning, he was taking the medication irregularly due to moderate local side effects (rush, skin burning). Patient 1 was re-educated about the aim of treatment, mode of application, and then continued the treatment regularly. After 3 months of follow-up, local side effects completely disappeared. In parallel, lowgrade fever and fatigue resolved. Inflammatory parameters normalized: CRP to 0.1 mg/dL (reference range: 0.8 mg/dL), SAA to 0.6 mg/dL (reference range: to 0.64 mg/dL) and stay within reference range after 24 months treatment. His kidney function and proteinuria are stable (creatinine level 1.7 mg/dL, proteinuria 2.0 g/day).

From the patient's perspective: during the 2-year follow-up despite initial doubts related to local reactions after administration of the preparation, we observed a very good compliance with the treatment principles. The applied targeted therapy not only resulted in the normalization of inflammatory parameters, but also eliminated the troublesome symptoms of excessive fatigue and the patient returned to his work on the farm. Currently, he feels free from burden of symptoms and from his point of view can give financial and personal support for his family members.

Patient 2 has started the treatment with IL-1 blocker (anakinra). Rapid response was observed with remission of symptoms within 2 days and normalization of lab results after 7 days (CRP 0.8 mg/dL; SAA 7.3 mg/dL). There were no flares during next 18 months of follow-up.

Patient 3 and Patient 4 remain asymptomatic and have currently normal laboratory results. They do not need chronic pharmacological treatment at present and are under thorough medical control.

#### **4. Discussion**

We present results of diagnostics that included NGS sequencing in a patient with kidney AA amyloidosis and all his children. The clinical presentation of the adult male patient was suggestive for AIDs, but not specific for a particular type of AID, including TRAPS, according to the clinical criteria acknowledged at the time of patient's admission [20]. Predominant clinical symptoms were episodes of fever and severe abdominal pain in childhood, arthralgia and fatigue in adulthood, and AA amyloidosis as a long-term complication. Patient's family history was negative, as well as the medical history in respect of migratory rash and periorbital edema—the more specific indicators for TRAPS [21]. According to the literature, the experts asked to indicate signs and symptoms regarded the most helpful in their practice for the diagnostics of TRAPS cited the following: recurrent long-lasting fever episodes, positive family history, periorbital edema, abdominal pain, myalgia, cutaneous rash, arthralgia, and monocytic fasciitis [22]. The onset of the disease in the reported male patient, as in most cases of TRAPS, was in his childhood, but at the time of diagnosis he presented the pattern typical for a chronic disease without inflammatory, acute flares. Notably, in his case it took approx. 29 years from the onset of the disease till the confirmation of diagnosis. Moreover, the proband developed AA amyloidosis and kidney failure, but it occurred quite early, in his twenties, while Lachman et al. found that the median age of developing of amyloidosis in TRAPS population was 43 years [5].

In clinical practice, there is a challenge to appropriately qualify the sporadic cases of AA amyloidosis for further genetic evaluation. Generally, when the clinical symptoms presented by the given patient are adherent with the diagnostic criteria for a specific AID (e.g., TRAPS), the genetic method of choice should be a targeted sequencing of the respective gene by the Sanger method. In the adult patient with AA amyloidosis reported hereby, an initial attempt was made of a targeted sequencing of selected exons of the *MEFV* gene, which proved unsuccessful. In the last decade, due to application of the next-generation sequencing (NGS), the genetic diagnosis in patients with AIDs has greatly improved and remarkable progress has been made in the genetic characterization of the undiagnosed

patients and the sporadic cases [23]. Importantly, TRAPS is genetically heterogeneous. More than 140 *TNFRSF1A* variants have been recognized up to date [24]. *TNFRSF1A* gene comprises 10 exons. Most of the variants are single-nucleotide substitutions (95%), but deletions and insertions have also been reported. Most sequence variants lie within exons 2 to 4 and result in amino acid substitutions which disrupt important cysteine-cysteine disulfide bonds within the extracellular domain (cysteine-rich domains, CRDs). A single splicing mutation also perturbs the first CRD due to the insertion of 4 amino acids. The most common are low-penetrance variants R92Q and P46L. Another common variant is T50M. Generally, mutations that result in cysteine substitutions lead to higher penetrance of the clinical phenotype (93% versus 82% for non-cysteine residue substitutions), and increase the probability of developing amyloidosis (24% versus 2% for no cysteine residue substitutions) [2].

Hereby, to establish the diagnosis, we performed WES with subsequent analysis of genes connected with autoinflammatory diseases. As a result, we identified a novel *TNFRSF1A* variant of in-frame insertion type that putatively influences three cysteine bridges and the structure of TNFR1 protein leading to its dysfunction or loss of function. The variant was confirmed by Sanger analysis. To the best of our knowledge, this variant has not been reported up to date. Thus, the important question is whether this novel genetic variant is pathogenic. Notably, it was found in one allele, which corresponds to the fact that TRAPS is an autosomal dominant disease. Clinical arguments for its pathogenicity are that the same genetic variant was found in three symptomatic relatives (i.e., daughters). However, clinical spectrum varies among family members and leads to personalized management. Therefore, further research laboratory tests, e.g., in the cell culture models, are necessary to respond the question of pathogenicity with acceptable precision.

Management of TRAPS should be adjusted case by case. It can involve corticosteroid therapy, NSAIDs during flares or a treatment based on biological agents, mainly TNF receptor-IgG1 Fc fusion protein etanercept [25] or interleukin 1 inhibitors [9]. Current data indicate that anti-IL-1 compounds are the most effective drugs in patients with pathogenic variants [26]. Such treatment eliminates the clinical symptoms and inhibits progression of AA amyloidosis. Indeed, the efficiency of anti-IL-1 agents in TRAPS treatment has been proved. Anakinra, a recombinant and slightly modified version of the human interleukin 1 receptor antagonist protein or canakinumab, a human monoclonal antibody targeted at interleukin-1 beta can be used. In 47 TRAPS patients from the US, the European Union, and the eastern Mediterranean, treatment with anakinra versus anti-TNF agents as the first biologic therapy led to significantly higher clinical and biochemical responses [27]. In one adult with amyloidosis-related renal failure, anakinra led to the disappearance of manifestations of TRAPS and decrease of laboratory abnormalities, including proteinuria [28]. Moreover, this treatment is considered safe, as in the presented case the side effects were local and self-limiting. Canakinumab was effective in controlling and preventing fever flares in patients with TRAPS, colchicine-resistant FMF, and MKD [29,30]. In contrast to anakinra, which is administered daily, canakinumab can be given every 4–8 weeks. The limitation of its use is the high cost, and that at present time it is not reimbursed in Poland.

As mentioned above, management of TRAPS and qualification for biologic treatment should be personalized. In this study, three additional family members of the patient are symptomatic and have undergone diagnostic workout, but based on their symptoms and the estimated risk of end-organ damage, the goals and manners of management differ. The father (proband) is treated continuously with anakinra to stop the AA amyloidosis progression. His 7-year-old daughter received anakinra treatment before any damage accrual occurred, and in future it may be possible to pause the medication. Finally, two daughters are covered by watch-and-wait strategy, as flares were sporadic, and inflammatory markers are within reference range between attacks.

Our study has some limitations. We did not perform the functional studies on the newly identified variant. Moreover, the pathogenicity of the variant could be further supported by the assessment (e.g., by flow cytometry) of TNFR1 presence on the respective cells, and the serum concentrations of soluble TNFR1, TNF-α, and perhaps other proinflammatory cytokines. However, none these tests are routinely performed in the medical centers involved in the current study. Furthermore, given the information collected in this report, it would be preliminary to speculate what would be the pathophysiological and/or molecular mechanism(s) responsible for the pathogenicity of the newly identified variant. In theory, such effects may rely on the exaggerated activation of NF-κB pathway [18] and/or hyperresponsiveness to the proinflammatory stimuli [19], and then induction of subsequent inflammatory cascading. As mentioned, this should be further investigated in properly designed functional studies. Despite its limitations, our study supports the notion that in cases of AA amyloidosis of unknown origin, the monogenic AID, including TRAPS, should be considered.

#### **5. Conclusions**

TRAPS is a very rare disease and can be overlooked in differential diagnosis, especially if clinical picture is not specific. The diagnosis is usually delayed leading to organ damage with the most serious complications, i.e., the development of secondary inflammatory amyloidosis and kidney failure. In diagnosis, the crucial role is played by the genetic testing, preferably by NGS. Hereby, in an adult male with AA amyloidosis and his family members we identified a novel in-frame insertion-type pathogenic variant in one allele of *TNFRSF1A* gene that caused TRAPS. Setting up the correct diagnosis of TRAPS allows for the choice of a suitable treatment. Indeed, anti-IL-1 agents provide efficient and safe therapy that eliminates clinical symptoms and prevents the patients from the organ damage, which is exemplified in the current report.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2077-038 3/10/3/465/s1, Figure S1: Next-generation (whole exome) sequencing and alignment visualization using Integrative Genomics Viewer (IGV), Table S1: Genes connected with inborn errors of immune system analyzed by NGS in 33-years-old man with AA renal amyloidosis.

**Author Contributions:** Conceptualization, J.Z., E.W.-S. and R.Z.; methodology, K.C.; software, A.S.- T. and P.S.; validation, M.K.; formal analysis, K.C. and B.W.-K.; investigation, B.W.-K. and E.H.; resources, P.S. and D.S.; data curation, K.C. and P.S.; writing—original draft preparation, J.Z. and E.W.-S.; writing—review and editing, R.Z.; visualization, K.C. and P.S.; supervision, L.P., K.J.-R. and E.B.; project administration, E.H. and D.S.; funding acquisition, R.Z and L.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The local Bioethics Committee of the Medical University of Warsaw waived the requirement for ethical permission due to the retrospective character of this noninvasive follow-up observation study (waiver No. AKBE/339/2019, 2 December 2019). All routine diagnostic and therapeutic procedures described within the study were carried out after obtaining a written informed consent from the patient.

**Informed Consent Statement:** We obtained a written informed consent from the patients/representatives for the publication of this report.

**Data Availability Statement:** The novel molecular variant was 184 submitted to the ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar; Submission ID: SUB6616942).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*2.6. Bronchoalveolar Lavage Cell Counting*

Bronchoalveolar lavage (BAL) cells were separated from BALF by centrifugation at 1000× *g* for 3 min. Then, the supernatants were collected for ELISAs, and the cell pellets were resuspended in PBS. Slides of BAL cells were processed using a CytoSpin (CytoSpin, Hanil Science, Gimpo, Republic of Korea) at 500 rpm for 5 min and stained by a Diff-Quik staining kit (Sysmex, Kobe, Japan).


*2.4. Additional Covariates*

Demographic factors including age, categorised as young adult (18–35 years), middle-age (36–50 years) and older-age (>50 years), and sex of the household reference person were self-reported. Additionally, two indicators of socioeconomic status were patterned by both exposure and outcome: educational attainment categorised as 'None, GCSE or equivalent (≤11 years)', 'A-level (12–13 years)', 'Vocational qualifications (12–13 years)', 'Degree or higher (>13 years)' or 'Missing', and equivalised household income categorised as '£14,999 or below', '£15,000–£24,999', '£25,000–£34,999', '£35,000–£49,999', '£50,000 and above' or 'Missing'. Missing covariates were examined across all exposure variables, with no significant differences in percentages across exposure levels found. As a

result, we included cases with missing covariate data in appropriate models to improve estimates and avoid case deletion (i.e., the missing indicator approach) [38].


*Article* **Implementation Framework for Energy Flexibility Technologies in Alkmaar and Évora**

**Nienke Maas 1,\*, Vasiliki Georgiadou 1, Stephanie Roelofs 1, Rui Amaral Lopes 2, Anabela Pronto 2 and Joao Martins 2**

Received: 30 September 2020; Accepted: 4 November 2020; Published: 6 November 2020

**Abstract:** As energy generation based on renewable resources does not always match energy consumption profiles, Positive Energy Districts (PEDs) should embody energy flexibility technologies to decrease possible negative impacts on existing grids due to, e.g., reverse power flows. As part of the EU H2020 Smart Cities and Communities project POCITYF, the cities Alkmaar (NL) and Évora (PT) aim to support the deployment and market uptake of such districts and in doing so demonstrate innovative and integrated technologies to enable flexibility in the energy system. This paper addresses implementation conditions for energy flexibility technologies that help cities to engender the expected impact and ensure replication of these technologies to other sites. It aims to guide both urban planners and technology solution providers through pitfalls and opportunities that can appear during the design and implementation of PEDs. Taking this into consideration, the RUGGEDISED innovation and implementation framework for smart city technology was taken as a starting point to describe and analyze the experiences in Alkmaar and Évora.

**Keywords:** governance; energy flexibility; positive energy districts; sustainable energy; smart city deployment

#### **1. Introduction**

The need for cities to become more sustainable is high and several definitions of smart cities refer to this need. A definition of a smart city is "a sustainable and efficient City with high Quality of life that aims to address Urban challenges (improve mobility, optimize use of resources, improve Health and safety, improve social development, support economic growth and participatory governance) by application of ICT in its infrastructure and services, collaboration between its key stakeholders (Citizens, Universities, Government, Industry), integration of its main domains (environment, mobility, governance, community, industry, and services), and investment in Social capital" [1]. However, retrofitting existing environments remains challenging [2]. Positive Energy Districts (PEDs) play an important role in more liveable and sustainable future cities. PEDs are districts producing energy from local and distributed renewable energy sources, presenting generation surplus over a specific balance period (typically one year) that may be transferred to areas outside a PED's boundaries [3]. One definition of PEDs by JPI Urban Europe (2019) refers explicitly to the active management of energy flows: "PEDs are energy-efficient and energy-flexible urban areas or groups of connected buildings which produce net zero greenhouse gas emissions and actively manage an annual local or regional surplus production of renewable energy" [4].

The large deployment of distributed generation based on renewable energy (RE) can increase the complexity of grid management and operation due to several factors (see, for instance, the impact of reverse power flows on distribution transformer aging [5]. Increased energy flexibility in the existing energy systems is therefore a crucial mechanism to delay costly and overdesigned adaptation of the grid infrastructure itself [6]. In this context, technology can be of great help in linking resource efficiency and flexibility in energy supply and demand with innovative, inclusive and more cost-effective services for citizens and businesses. Such technologies can integrate infrastructures like smart grids that have been piloted in several cities in FP7 and H2020 projects [7].

In Alkmaar (Netherlands) and Évora (Portugal), innovative and integrated technical solutions are being implemented in order to support the deployment and market uptake of PEDs. This research and the pilots in Alkmaar and Évora are being conducted within the EU H2020 Smart Cities and Communities project POCITYF [8]. The project started in October 2019 and includes the demonstration of solutions of a high technology readiness level (TRL ≥ 6) [9] for achieving flexible and efficient use of electricity in contexts with different climatic conditions and regional characteristics (technical, financial, social and legal). Alkmaar and Évora are proving grounds for innovative and integrated technical solutions for buildings and districts in which energy management systems are implemented to increase flexibility. Starting points are lessons learnt from pre-pilots at other sites, i.e., locations and buildings where individual technologies have been implemented before. The second step, which is dedicated to demonstration activities, combines technologies toward integrated systems at the building, block and district levels in the areas of Alkmaar and Évora. The third step refers to replication of these integrated systems in other selected areas of Alkmaar and Évora and in the six fellow cities: Granada (Spain), Bari (Italy), Celje (Slovenia), Újpest in Budapest (Hungary), Ioannina (Greece) and Hvidovre (Denmark).

Starting from the RUGGEDISED innovation and implementation framework developed in the EU H2020 RUGGEDISED project [10], this paper addresses implementation conditions for energy flexibility technologies in Alkmaar and Évora in order to support the achievement of expected impacts and ensure replication of these technologies within and beyond POCITYF. The technologies under consideration are ReFlex [11] for Alkmaar and flexibility control algorithms for Évora. Both aim at exploiting the energy flexibility provided by the available controllable devices (e.g., batteries or electric water heaters) at the building, block and district levels in order to achieve specific objectives (e.g., improve matching between renewable generation and energy demand or decrease peak loads).

The RUGGEDISED innovation and implementation framework is an analytical tool that helps city planners to assess important success factors in the implementation process of smart technologies well in advance. This framework focusses on smart city technologies in a broader sense without concentrating necessarily on energy flexibility technologies alone. Therefore, this paper aims to guide both urban planners and technology solution providers through pitfalls and opportunities that can appear during the design and implementation of a PED. It describes practical examples of implementation conditions seen or experienced in Alkmaar and Évora (or that were missing). This can be used to describe valuable lessons for the implementation of energy flexibility in future PEDs. This framework identifies both the implementation conditions that were of relevance and influence in either case of the two cities, and those conditions that did not play a role. In doing so, it provides insights in improving and adjusting projects and shifts the focus on conditions that matter. This paper discusses the application of the framework by presenting how it is being used in practice for the analysis of the implementation of PEDs in Alkmaar and Évora.

#### **2. Materials and Methods**

The RUGGEDISED framework [10] can be used to assess the implementation process of smart city projects and therefore "advises" what should be in place for successful implementation. In POCITYF, records of progress on pre-pilots and foreseen demonstration activities in Alkmaar and Évora have been taken, using questionnaires. These questionnaires focused on key technical components and specifications of the innovative solutions, on the demonstration sites (considering specific challenges related to the context), on problems and restrictions experienced and on lessons learnt

(e.g., technical improvements, energy savings or socioeconomic benefits). The project partners involved with each innovative solution took care of the respective questionnaires. These project partners work for different organizations, e.g., research institutes, governments, utilities and technology providers.

Despite common challenges, the cities of Alkmaar and Évora face city- and district-specific challenges due to divergent geography, geology, demography, climate and socio-economic and cultural characteristics. These characteristics mean that urban energy transition challenges are embarked upon from different starting points and perspectives, thus enhancing the complementarity of the POCITYF solutions. Évora represents South European cities, which generally show lower investments on reducing the footprint of their households and business sector but can enjoy an abundant solar potential. Alkmaar represents West European cities that are strongly dependent on gas for electricity and heating.

In more detail, the RUGGEDISED framework provides a useful base for analyzing success factors and hindering factors for the implementation of smart technologies. In POCITYF, innovative smart city solutions are implemented in order to achieve PEDs. This framework (see Table 1) was consolidated in the EU H2020 RUGGEDISED project and allows the analysis of suppressing and enhancing factors in implementation processes of smart solutions. Such an analysis is beneficial in designing successful implementation processes, in assessing the potential project impact and in selecting aspects that need further consideration for successful implementation.

**Table 1.** RUGGEDISED innovation and implementation framework: enhancing and suppressing factors [10].

The framework distinguishes three levels of impact that are needed in order to think beyond implementation of single solutions and consider the real impact of implementation, namely:

The implementation factors influencing the development of smart solutions are described per impact level. A division is made between hardware, software and orgware implementation factors. Hardware focuses on physical infrastructure topics such as energy storage, conversion and savings. Software refers to ICT, data-related factors and applications. Hardware and software must be supplemented with orgware, which refers to organizational and governance aspects such as stakeholder management, institutional and organizational arrangements and innovation platforms. Dividing impact levels and implementation factors in three categories allows for structuring the different factors that influence projects in different stages. Some factors help or hinder the realization and output of smart solutions, whilst others specifically impact the working together of multiple solutions to realize embedded outcomes. The framework shows that the importance of orgware implementation factors increases as the impact level rises. These orgware implementation factors, in turn, affect the upscaling and replication of smart solutions.

#### **3. Results**

One of the objectives of the POCITYF project is to deploy and validate smart energy management and storage solutions to optimize energy flows with the goal of maximizing self-consumption, reducing grid stress and valorizing flexibility services. To that end, innovative solutions to be demonstrated and replicated include several individual elements: e.g., low-temperature waste heat, innovative short- and long-term storage solutions, such as hydrogen fuel cells or electrical vehicles (EVs) coupled with stationary batteries, smart ICT solutions to interconnect the energy management system at the household, building and district levels, the virtual power plant (VPP) concept, thermal grid controllers or market-oriented building flexibility services.

Figure 1 illustrates how the individual elements corresponding to the various layers, from concrete, device-specific to abstract, domain-specific, are stacked on top of each other and interact to bring about the full implementation and impact of an integrated innovative solution. On the left side, the data and control flow is bidirectional for the full stacked solution to perform as per design, while on the right side, there is only sharing of information to gain insight into the system, evaluate different options and decide for further optimization and improvement steps. The loop is closed by feeding these insights back to the operational system in place.

**Figure 1.** From the concrete, device-specific layer to the abstract, domain-specific one.

Taking this into consideration, Tables 2 and 3 map the information associated with the technologies under analysis (i.e., ReFlex for Alkmaar and flexibility control algorithms for Évora) based on the impact levels and implementation factors that comprise the RUGGEDISED framework described in Section 2.

**Implementation Factor Explanation** Level 1 realization and output **Software** Privacy • Including privacy by design in order to meet both national and European standards and adhere to GDPR guidelines. In doing so, privacy concerns will not pose obstacles to implementation. Security • Taking cyber security issues into account as they may arise. Including privacy and security by design so that they will not become an obstacle for implementation and the implementation will meet the required national and European regulations and standards. It can be helpful to follow the eight principles of privacy by design [12]. Smart grid ICT • Internet of things software from ICT.eu links all electricity consumers per platform. The data are made accessible via the cloud, giving the energy supplier remote control. Via this platform, everything can be connected per household that uses or generates energy. User interfaces • Using future-proof standards (even with low maturity) to implement new use cases and future business models. **Orgware** Business models • Cost reduction of grid operations as a result of unlocking and exploiting flexibility. • New revenue streams potential from valorizing flexibility. • Analysis of pricing mechanisms and market liquidity has been carried out as well as a usability analysis. • Bundling multiple smaller devices to increase accessibility to the trade market. Data and data ownership • Handled according to the principle "as open as possible, as closed as necessary". Level 2 embedded outcomes of multiple smart solutions **Hardware** Communicating infrastructure • Continuous internet connection in order to communicate in real time between assets, the network and the energy market. Robustness of the system • Fast response to disconnection. • Backup facility. • Risk management: what goes wrong if the system is down or unavailable? Taking measures appropriate to the risks (system overload or just some missing data). • The placement of bidirectional charging stations is currently not a standard job. They are still far more expensive than standard charging stations, they need a reliable internet connection to function and during the City-zen project, problems with the hardware were encountered frequently. • The reliability of OT/IT connections through 4G and Wi-Fi communication networks used for VPP and V2G applications is lower than that of the grid itself. The reliability needs to increase to match the reliability of the grid before a VPP or V2G can be used as part of the vital infrastructure balancing loads on the LV grid.

**Table 2.** *Cont.*

**Table 2.** *Cont.*

**Table 2.** *Cont.*

#### **Table 3.** Mapping Évora.

**Table 3.** *Cont.*

**Table 3.** *Cont.*

#### **4. Analysis**

The RUGGEDISED framework allows for conducting an analysis of important implementation conditions in the cities of Alkmaar and Évora. This section presents the respective main observations.

(in time, budget and purpose). This results in the delivery of flexibility technologies that function (Level 1) but will not necessarily graduate to achieve Level 2 and Level 3 impact; as such, upscaling and replication are not induced. Single flexibility technologies are delivered but this does not result in energy flexibility as a whole or flexibility in smart city systems. At the community or neighbourhood level, subsidies are effective in reaching goals, but in order to achieve large-scale impact (Level 2 and Level 3), combined strategies and widespread collaboration are needed.

#### **5. Conclusions**

In this paper, the RUGGEDISED framework was applied as the starting point to evaluate the implementation conditions of energy flexibility solutions within the POCITYF project. Indeed, the selected framework can be leveraged to provide a first analysis of both the success and hindering factors for the implementation of smart technologies while taking a holistic approach that includes not only technological perspectives but also organizational and systemic ones.

The main recommendations and findings for implementing energy flexibility-related technologies at the PED level are summarized below.

loop enables deeper understanding of the sources of error, that can either be found in the control (use) or in the characterization function. For acceptance of energy flexibility, it is important to close this loop. Insights delivered via tracking key performance indicators (KPIs) are needed both on rational arguments (economic and energy metrics) and on perception arguments (e.g., satisfaction of citizens).

The experiences of Alkmaar and Évora result in an easy-to-use methodology for cities to deal with energy flexibility technologies and create optimal conditions for their successful implementation and integration. In the coming period, demonstration of the related energy flexibility technologies is being carried out in both cities at the block and district levels. Starting from the analysis, progress is to be tracked especially focused on achieving Level 1 and moving forward to higher impact levels. This effort will also support the replication process not only in other areas within Alkmaar and Évora but also the so-called "fellow" cities participating in the POCITYF project.

#### **6. Practitioners Review by Roel Massink MSc**

The authors of this paper asked me to provide my view on the methods and results of this research paper in my capacity as Project Coordinator for the H2020 Smart City Lighthouse project IRIS and innovation manager for the Municipality of Utrecht. I am happy to fulfil this task and I thank the authors for the carefully presented research results based on actual demonstration. This review is based on the experiences collected in the IRIS Smart Cities project, a similar project to RUGGEDISED and POCITYF. In IRIS, an integrated project approach is demonstrated: in the Kanaleneiland district (Utrecht, The Netherlands), near zero-energy-efficient building retrofitting is connected to the development of a smart energy and mobility system integrating PV panels with stationary and V2G storage. The smart solutions in IRIS Smart Cities Utrecht mostly all fall within Level 2 of the RUGGEDISED implementation framework. Below, some general reflections based on these experiences toward the RUGGEDISED implementation framework and the conclusions of the paper can be found.

The structure of the RUGGEDISED framework is very much in line with what is happening in practice in the IRIS Smart Cities project and further development of the framework in practice is appreciated. Within the IRIS Smart Cities project, the smart solutions are implemented along transition tracks and structured through a maturity assessment in the following categories: (1) pre-pilots: solutions that have been tested on a small scale in a pilot project; (2) integrated solutions: multiple smart solutions that are demonstrated as integrated solutions at a larger scale at the demonstration site of the project; and (3) replicated solutions: through either the copying of a successful integrated solution to other sites or by upscaling of the integrated solution in the same city or region. This categorization provides the IRIS partners and stakeholders a framework and a roadmap for all smart solutions to be developed, demonstrated and scaled-up. The RUGGEDISED framework is valuable and will

offer practitioners at public authorities, grid operators, solution providers and knowledge institutes a coherent overview of what is important for implementation. However, below I have some suggestions in view of the practical applicability of the RUGGEDISED framework. First, smart solutions are integrated solutions by nature. To ensure that the smart solutions (that are realized in isolation; Impact Level 1) meet the requirements of an embedded outcome of multiple smart solutions (Impact Level 2), it is suggested that an integrated approach with all implementation factors is taken into account. To ensure such an integrated (and systemic) assessment throughout the development of smart solutions, an iterative execution of the implementation framework could be proposed. This means that at Level 1, the implementation factors of Level 2 and Level 3 are also considered. Assessment of implementation factors in Level 2 and Level 3 includes more detailed and specific information once the development of smart solutions progresses.

The following three reflections are supportive of including higher-level implementation factors at the start of the pilot projects.

The paper highlights the importance of aligning partner goals with pilot project aims. This should be seen as a conditional factor for achieving progress at the different impact levels. An example from IRIS Smart Cities Utrecht is the development of a bidirectional charging ecosystem for grid flexibility and mobility services. Currently, this integrated solution consists of multiple smart solutions (bidirectional charging infrastructure, bidirectional enabled electric vehicles, stationary battery storage, energy management system) that are being connected to provide embedded outcomes (Level 2). The current state of development is a result of careful stakeholder management from the start of the pilot projects (already pre-IRIS). Companies, knowledge institutes, grid operators and public authorities aligned their roadmaps and activities in pilot projects under the lead of an ambitious SME (Level 1) and now this is leading to embedded outcomes in the IRIS demonstration project (Level 2). Furthermore, scaled adoption in public procurement documents of electric charging infrastructure now paves the way for city-wide flexibility services of electric vehicles (Level 3). An important success factor was/is stakeholder alignment from the start of the pilot projects. Based on this experience, it is argued that stakeholder management (or rather stakeholder alignment) should take position already at Level 1 to ensure that partner roadmaps are aligned to move the smart solution to Level 2 and 3.

The paper also points towards the requirement of end-user involvement (or co-creation) in the development of smart solutions and could argue for the inclusion of a new implementation factor in the RUGGEDISED framework. This could be "end-user satisfaction" or similar. This is a valid point; often smart solutions are hampered because end-user needs were not considered well enough in the original approach. A design-thinking (or another systemic) approach could offer smart solution developers tools to better involve end-users. A practical notion that requires attention here is the use of grant subsidies in this innovation framework (as the authors also refer to in moving from Level 1 to Level 2). It is recommended that subsidy programs, grant applicants and consortia put more attention into making end-user involvement more explicit in call texts and subsequently also allow more flexibility in the implementation of the grant project based on changes offered by end-users.

Next to this, the paper explains the requirement of a legal framework that supports the transition from pilot projects to scaled solutions. This is true as well for the IRIS Smart Cities project. Especially within the field of smart energy projects or services like peer-to-peer, a supportive legal framework is needed for scaled adoption. The exploitation of grid flexibility services is hampered by legal constraints. Research from the IRIS project shows that bidirectional charging services are discouraged because of double energy taxation (for each charging and discharging cycle, energy tax needs to be paid on either the stored or consumed kWh). This significantly hampers the realization of profitable business models and commercial scaling of these integrated smart solutions. Therefore, it could be argued that regulation/the legal framework is already introduced as an implementation factor in Level 2 of the framework to ensure that, in time, legal constraints are targeted in a concerted action by smart cities.

Finally, what the paper does not explain but what is highly valuable for practitioners is guidance on how the implementation framework leads into an implementation process. The presented implementation framework provides an assessment of implementation factors related to smart solutions but does not directly translate the assessment into implementation guidelines. Further guidance on the implementation pathway resulting from the assessment could support practitioners in applying this framework more easily.

**Author Contributions:** Conceptualization, N.M., V.G. and S.R.; methodology, N.M. and S.R.; validation, N.M., R.A.L. and J.M.; formal analysis, N.M., V.G., S.R., R.A.L. and J.M.; investigation, N.M., V.G., S.R., R.A.L., A.P. and J.M.; resources, S.R. and R.A.L.; writing—original draft preparation, N.M., V.G., S.R., R.A.L., A.P. and J.M.; writing—review and editing, N.M., V.G., S.R., R.A.L. and J.M.; visualization, V.G.; supervision, N.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by POCITYF, (FP7 grant agreement N◦ 864400). And The APC was funded by EERA Joint Program Smart Cities.

**Acknowledgments:** This paper is based on information collected during the initial phase of EU H2020 Smart Cities and Communities project POCITYF (grant agreement N◦ 864400). The authors would like to express their gratitude to Roel Massink MSc, coordinator of the H2020 Smart City Lighthouse project IRIS, for reviewing the methods and results of this research and for all the suggestions provided.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

*Energies* **2020**, *13*, 5811

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**4.2 Side effects of thiamin**

Adverse reactions to thiamin administration have been reported as reactions at the injection site, but their frequency is unknown. Other side effects can be diaphoresis, pruritus, skin sclerosis (at the injection site after IM administration), urticaria, nausea, bleeding (in the digestive tract). For hypersensitivity side effects, reported anaphylaxis (after IV administration), angioedema, hypersensitivity reactions (following IV administration). Side effects of intravenous thiamin are rarely reported. A prospective study by Wrenn in 989 samples given thiamin 100 mg IV found adverse reactions in the form of minor reactions, which were transient local irritation in 1.1% and pruritus in 0.0093% of patients. Thiamin hydrochloride can be given intravenously without problems. An intradermal test dose before administration is not required unless the patient had a previous allergic reaction [12]. Local side effects for larger doses can be minimised by slow administration into a larger and more proximal vein. Thiamin should be administered before parenteral glucose solutions to prevent Thiamin deposition as a symptom of acute thiamin deficiency in malnourished patients [11].

#### **Table 1.**

*Recommended* dietary allowance *(RDA) of thiamin [2].*

Alcohol consumption can interfere with intestinal absorption of vitamin B1, and chronic alcoholism leads to *Wernicke-Korsakoff syndrome* (WKS) [2]. Intoxication may occur with ingestion of more than 3000 mg thiamin in the long term. Based on animal research, thiamin lethal dose/LD50 are 8224 mg/kg, while the LD50 in rats are 3710 mg/kg [13]. Sporadic anaphylactic reactions have been reported. Some researchers suggest that intravenous thiamin should be administered in a resuscitation facility. However, due to the life-threatening nature of WKS, EFNS (European Federation of Neurological Society) guidelines recommend starting treatment immediately, even in the absence of facilities for resuscitation [14].

### **4.3 Thiamin and brain metabolism**

Thiamin has an essential role in brain metabolism. Nerve cells use glucose as the primary fuel in producing energy. Glucose reaches brain tissue by diffusion across the blood–brain barrier. Around 30% of glucose absorbed by the brain undergoes complete oxidation through the Krebs cycle [15]. Various mechanisms contribute to selective brain lesions observed in WKS and thiamin deficiency. Recent evidence of early microglial activation and increased production of free radicals suggests that oxidative stress processes play a vital role in brain cell death associated with thiamin deficiency. Recent studies in animal models of WKS demonstrated changes in thiamin-dependent enzymes in the brain and suggested that changes in these enzyme activities may result in neuronal death, characteristic of this syndrome [16].

Thiamin is needed as an enzyme cofactor essential for brain metabolism, and around 80% of total thiamin is in the neural tissues [10, 17]. In addition to its co-enzymatic function in metabolism, thiamin also has a structural role [18, 19]. Thiamin affects membrane structure and function, including axoplasmic, mitochondrial, and synaptosomal membranes, which act against agent-induced cytotoxicity and improve membrane location [20, 21]. Thiamin also intervenes in synaptic transmission and plays a role in cell differentiation, synapse formation, axonal growth, and myogenogenesis [2].

#### **4.4 Thiamin as a novel treatment for sepsis**

Thiamin deficiency is common in critically ill patients and correlated with increased mortality in some cases. In addition, its levels are depleted throughout the illness, and administration of thiamin during critical illness can improve organ dysfunction [22]. Predisposing factors to thiamin deficiency result from several associated problems associated with nutritional disorders and other accompanying diseases. Several conditions can reduce thiamin levels, such as impaired carbohydrate metabolism, increased metabolic requirements for parenteral or enteral nutrition, diuretics, and haemodiafiltration. Several studies have found the presence of thiamin deficiency in critically ill patients. Thiamin deficiency is associated with poor prognostic outcomes [23].

A cohort study in Australia with 129 patients found no association between plasma thiamin concentrations with systemic inflammation and mortality in critically ill patients. In addition, it also shows that level of thiamin intake in patients who had not received its supplementation before ICU admission did not differ between patients who died and those who survived, 264 compared 268 nanomol/L (normal value: 190–400 nanomol/L). Besides that, only a weak correlation was found between thiamin levels and disease severity index. A study had investigated the correlation of thiamin, APACHE II, SOFA score, maximum SOFA score, SOFA delta (DSOFA), and CRP. However, the correlation is not statistically significant [24].

Thiamin deficiency can also be observed in septic shock patients, occurring in 8.5–72% depending on the cutoff value used to determine thiamin deficiency [4, 22, 25, 26]. Lack of thiamin reduces pyruvate access to the Krebs cycle, increasing lactate production as it converts metabolism to anaerobic [23].

A prospective observational study examined the association between thiamin levels and lactic acidosis in 30 septic shock patients and found no correlation between these variables. However, after excluding patients with abnormal liver function, a significant negative correlation was found between thiamin concentrations and lactic acidosis (r = −0.53, P = 0.01). This finding implies a potential relationship between thiamin levels and lactic acidosis in septic shock patients with normal liver function. Thus, by reducing pyruvate dehydrogenase complex activity, thiamin deficiency contributes to an increase in lactic acid in septic patients [26].

Parenteral administration of thiamin 250 mg once daily for 3–5 consecutive days is recommended to treat thiamin deficiency. Slow intravenous administration of thiamin diluted in isotonic NaCl or 5% dextrose is also safe. However, there is no consensus on the optimal daily dose of thiamin, its formulation, and duration of treatment [14]. The half-life of unphosphorylated thiamin blood is 96 hours. Therefore, two or three daily doses can achieve better concentrations in the brain than a single daily dose. In patients who do not consume alcohol, a daily intravenous dose of 100 or 200 mg is sufficient to meet thiamin requirements. However, alcoholic patients with WKS may require doses as high as 500 mg three times daily [14].

A clinical trial of *ascorbic acid and Thiamin effect in septic shock* (ATESS) conducted in South Korea compare outcomes of a combination of ascorbic acid (IV 50 mg/kg, maximum dose per dose of 3 g) and thiamin (200 mg) every 12 hours for two days with placebo groups on 111 subjects. The results showed no significant differences in SOFA scores and organ function but found an increase in serum levels of vitamin C and thiamin [27].

This finding is in contrast to another clinical study in 94 patients who received a combination of 1500 mg of vitamin C IV q6, 200 mg of thiamin IV q12 for four days or until discharge from ICU, and 50 mg of hydrocortisone IV q6 (with the optional alternative of 50 mg bolus, followed by continuous infusion of 200 mg in 24 hours) for four days. Thiamine administration had significantly reduced the progression of organ dysfunction and mortality in patients with severe sepsis and septic shock. However, the research design had weaknesses include small study size, pre-and post-study design, single-centre, absence of blinding, and presence of three simultaneous interventions limiting the generalizability of the conclusion. Although it can help invent new hypotheses in future, this study is still not strong enough to produce a change in clinical practice [22].

#### **5. Conclusion**

Thiamin plays a vital role in cell metabolism. The administration of thiamin supplementation should be considered adjunctive therapy in critically ill patients as it may improve their outcomes. Further research should be developed to determine the optimal dosage and timing to achieve the maximum effect.

*Thiamin (B1) and Its Application in Patients with Critical Condition DOI: http://dx.doi.org/10.5772/intechopen.99626*


*1.2. Magnetic Pulse Welding*

In MPW, the sudden discharge of a capacitor generates very high electric currents through a coil, leading to a very high magnetic field pressure that accelerates toward one another the parts to be welded. Welding of a wide range of similar and dissimilar materials in different MPW configurations have been reported. For example, Okagawa and Aizawa [7] proposed a parallel MPW configuration that led to seam welds between 1 mm thick aluminum sheets. They experimentally studied the seam weld shearing strength and observed its dependence on the kinetic energy of the sheets before the collision and magnetic pressure after the collision. Watanabe et al. [8] obtained lap joints for aluminum flyer plates and iron, nickel, and copper base plates. They reported that exceeding a certain level of discharge energy resulted in a decrease in amplitude and wavelength of material interflow at the weld interface. Lee et al. [9] produced lap joints between 1.2 mm thick aluminum flyer plates and 1 mm thick low-carbon steel plates using MPW. They observed a slight flattening of the grains of aluminum plates in the vicinity of the weld after impact, but no change was apparent in the grain structure of the steel plates. Their nano-indentation hardness tests revealed increased hardness in the intermediate layer between the plates. Ben-Artzy et al. [10] showed that, in tubular MPW joints, reflected shock waves generate interference waves along the weld interface, and that the wavelength of interference waves is proportional to the free path of the shock wave propagation (the first path between front face and back surface) on the interior side of the weld. Göbel et al. [11] made a comparison between the physics involved in EXW and MPW. They reported that in MPW the impact velocity and angle changes along the weld, and that wave creation is possible at lower velocities in MPW compared to EXW. This allows for different welding windows (in terms of impact angle and velocity) in MPW and EXW. Raoelison et al. [12] performed an Eulerian simulation of MPW assuming a linear flyer velocity distribution having a mean value of 600 m/s for aluminum workpieces. They predicted thermomechanical material flow in the form of particle jetting. Cui et al. [13] proposed a method for MPW of 1.4 mm thick carbon-fiber-reinforced plastic and 1 mm thick aluminum tubes. They numerically imitated the MPW process using a coupled electromagnetic and mechanical simulation with constant stress elements.

### *1.3. Vaporizing Foil Actuator Welding*

In VFAW, a sudden capacitor-discharge generates a very high current through a thin conductor foil, vaporizing it almost instantaneously. This creates a very high-pressure plasma, which accelerates the flyer plate towards the target plate. The technique was introduced by Vivek et al. [14] in 2013. They successfully welded copper to titanium, copper to steel, aluminum to copper, aluminum to magnesium, and titanium to steel. Moreover, they quadrupled the weld strength of the titanium/steel combination by introducing a thin nickel interlayer. Hahn et al. [15] attempted an experimental comparison of VFAW and MPW and achieved success only for VFAW. Using the same charging energies of the pulse generator, with VFAW they reached velocities three times those for MPW. Vivek et al. [16] demonstrated two VFAW experiments to join copper sheets with 3 mm thick zirconium-based bulk metallic glass (BMG). In one experiment, the 0.5 mm thick copper sheet was directly launched towards the BMG, resulting in a straight welded interface. In the other experiment, a 0.5 mm thick titanium sheet was first launched towards a 0.25 mm thick copper sheet, consequently launching it towards the BMG, which resulted in a wavy weld interface and no devitrification of the BMG. Nassiri and Kinsey [17] created 2D simulations for VFAW of 2 mm thick flyer and 3 mm thick base plates of aluminum using ALE and SPH methods. In both methods, an initial constant flyer velocity and impact angle were assumed. While the SPH method was able to simulate material jetting, it was found to be less accurate compared to ALE.  They validated the ALE and SPH simulation results through VFAW experiments. ALE was deemed incapable of mimicking material vorticities. Chen et al. [19] machined slanted grooves in 6.3 mm thick steel plates at different angles ranging from 8 to 28 degrees and joined them to 0.508 mm thick aluminum flyers using VFAW. They used this technique to control the impact angle, and thus obtained various morphologies at the weld interface. In a separate study [20] that implemented this procedure, aluminum flyer plates of the same thickness were welded to 1.905 mm thick titanium plates. Gupta et al. [21] applied the same technique and similar foil thicknesses in VFAW of copper to titanium, and vice versa. They created an explicit thermomechanical Eulerian simulation with no slip in contact of Eulerian parts but again assuming a constant initial flyer velocity. It was shown that for capture of interfacial waves, sufficient mesh refinement must be implemented. Groche et al. [22] designed an experiment for process window acquisition in HVIW. They used 2 mm thick aluminum workpieces and reached total normal impact velocities up to 262 m/s (131 m/s for each workpiece). It was shown that in cases where the collision point velocity exceeds the speed of sound in aluminum, jetting would not occur, resulting in no bonding.


General Perception of Liposomes: Formation, Manufacturing and Applications

*Christian Isalomboto Nkanga, Alain Murhimalika Bapolisi, Nnamdi Ikemefuna Okafor and Rui Werner Maçedo Krause*

### **Abstract**

Liposomes are currently part of the most reputed carriers for various molecular species, from small and simple to large and complex molecules. Since their discovery, liposomes have been subject to extensive evolution, in terms of composition, manufacturing and applications, which led to several openings in both basic and applied life sciences. However, most of the advances in liposome research have been more devoted to launching new developments than improving the existing technology for potential implementation. For instance, the evolution of the conventional lipid hydration methods to novel microfluidic technologies has permitted upscale production, but with increase in manufacturing cost and persistent use of organic solvents. This chapter intends to present general concepts in liposome technology, highlighting some longstanding bottlenecks that remain challenging to the preparation, characterization and applications of liposomal systems. This would enhance the understanding of the gaps in the field and, hence, provide directions for future research and developments.

**Keywords:** phospholipids, soybean lecithin, liposome composition, manufacturing methods, characterization techniques

#### **1. Introduction**

Liposomes are artificial lipid-based bilayered vesicles. They were firstly discovered and described in 1965 by Bangham et al. [1] as swollen phospholipid systems, namely Banghasomes. A few years later, the structural description of liposomes was unveiled as small devices made of one or more closed phospholipid bilayers. Due to the diversity of particle sizes, from 20 nanometers to several micrometers, liposomal vesicles are considered as either nanoparticles or microparticles endowed with the ability to encapsulate materials of various nature and polarity [2, 3].

Up until now, liposomes have shown huge promise as potential vehicles for biologically active compounds in cosmetic and pharmaceutical industries. These applications have been extended to food and farm industries, where unstable substances such as antioxidants, flavors and antimicrobials have been explored for liposomal encapsulation. Across all these areas of application, liposomes have been deemed to be the most successful delivery systems due to their multiple advantages. These include high biocompatibility and biodegradability, low toxicity and poor immunogenicity, improved drug solubility and controlled distribution, as well as

the ability of performing surface modifications for targeted, extended and sustained release. Currently, there are several liposomal formulations that are clinically established for the treatment of various diseases, such as cancer, fungal and viral infections; and many more have reached advanced phases of clinical trials [4, 5].

Although liposomes have shown some success in drug product development, the limitations identified in liposomal technology have remained almost stagnant over decades. The most common disadvantages of liposomes arise partly from poor stability under shelf and in vivo conditions. This is mostly due to potential lipids oxidation and hydrolysis, leakage and loss of hydrophilic cargoes, as well as particles fission and fusion. To date, some of these problems can be circumvented by playing around formulation adjuvants, such as anti-oxidants, or post-preparation processing, such as freeze-drying [4, 6, 7].

While describing broadly the current perception of liposomes, regarding production, evaluation and applications; this chapter intends to highlight the longstanding bottlenecks that remained overlooked and challenging to product development and implementation. This would increase the understanding of the gaps in the field and provide future directions to new openings for improvements in liposome technology.