**5. Anesthesia for urological laparoscopic surgery**

Laparoscopic procedures in urology cover both oncological surgery like nephrectomy, prostatectomy, cystectomy and nononcological surgery like pyeloplasty. Laparoscopic surgery has found wide applications in urological surgery with the developing technol‐ ogy. After laparoscopic surgery, some complications due to pneumoperitoneum began to occur more frequent.

#### **5.1. Preoperative considerations**

An anesthetic plan is developed based not only on the patient's physical status determined by the assessment but on how the patient will tolerate pneumoperitoneum and body posi‐ tion during the surgery. Some factors like obesity and Trendelenburg level may increase the intraabdominal pressure during the laparoscopic operations. These factors should be con‐ sidered, when anesthetic management is planned. Difficult airway, cardiopulmonary status, allergies, medications and comorbid conditions are important issues for patients undergoing laparoscopic surgery. Especially, decision of laparoscopic surgery should be considered care‐ fully in patients with advanced respiratory disorder because of the high risk of anesthesia.

#### **5.2. Intraoperative considerations**

*4.2.1. Preoperative consideration*

62 Current Topics in Anesthesiology

*4.2.2. Intraoperative consideration*

stretch of the spermatic cord.

*4.2.3. Choice of anesthesia*

*4.2.4. Complications*

occur more frequent.

**5.1. Preoperative considerations**

erence, one technique may be more appropriate.

urological procedures. The sensorial block level must be Th10.

**5. Anesthesia for urological laparoscopic surgery**

These procedures generally do not require any particular anesthetic technique, depending upon the procedure, the medical condition of the patient and patient's and/or surgeon's pref‐

Routine monitorization is advised. During the varicocelectomy, bradycardia can occur due to

Many of these procedures are ambulatory, performed in cystoscopy suites with a rapid turn‐ over of patients and the anesthetic choice must also consider these concerns. Evaluation of the lower urinary system tract is often performed by the urologist with a flexible cystoscope. This procedure generally performed by the urologist with local topical anesthesia applied to the inside of the urethra as it does not require full bladder distention. If patient could not tolerate pain, the procedure must be performed under monitored anesthesia care with sedation [53]. Neuraxial anesthesia has been long considered the anesthetic technique of choice for these

During the varicocelectomy, bradycardia can occur due to stretch of the spermatic cord.

Laparoscopic procedures in urology cover both oncological surgery like nephrectomy, prostatectomy, cystectomy and nononcological surgery like pyeloplasty. Laparoscopic surgery has found wide applications in urological surgery with the developing technol‐ ogy. After laparoscopic surgery, some complications due to pneumoperitoneum began to

An anesthetic plan is developed based not only on the patient's physical status determined by the assessment but on how the patient will tolerate pneumoperitoneum and body posi‐ tion during the surgery. Some factors like obesity and Trendelenburg level may increase the intraabdominal pressure during the laparoscopic operations. These factors should be con‐ sidered, when anesthetic management is planned. Difficult airway, cardiopulmonary status, allergies, medications and comorbid conditions are important issues for patients undergoing laparoscopic surgery. Especially, decision of laparoscopic surgery should be considered care‐ fully in patients with advanced respiratory disorder because of the high risk of anesthesia.

Pneumoperitoneum and patient positioning impede normal respiratory mechanics. Placement of an endotracheal tube allows the ventilator to supply the work necessary to breathe. Gastric secretions are commonly seen in the oropharynx or on the face of patients at the end of sur‐ gery. The placement of an arterial line may be indicated if the patient's medical condition war‐ rants closer blood pressure monitoring nasogastric tube decompression of the stomach and Foley catheter drainage of the bladder is the basic procedure for most urologic laparoscopic surgeries. Hypothermia is common beginning with the disruption of thermal regulation due to anesthesia.

#### **5.3. Choice of anesthesia**

Most common anesthetic plan is general anesthesia. General endotracheal anesthesia is cho‐ sen to counter the adverse conditions created by the pneumoperitoneum, patient positioning and surgical time. If general anesthesia is contraindicated, high level sensorial block (Th4) can be performed.

#### **5.4. Complications**

Anesthetic complications are addressed through that prism: anesthetic strategies to mini‐ mize hemodynamic changes due to pneumoperitoneum and patient position. Increasing the intrathoracic blood volume improved hemodynamic function in all body positions with pneumoperitoneum. Fluid management is the most important element for minimizing pneu‐ moperitoneum side effects [54].

Most common observed complications of laparoscopic surgeries are swelling of the face, eye‐ lids, conjunctivae and tongue along with a plethoric color of venous stasis in the head and neck. Although facial edema is common, but laryngeal edema may prevent the extubation of patient and can cause delay extubation in 5% of patients [55, 56].


**E-Learning Organizational Infrastructure**

**1**

Snježana Babić

*Croatia*

**Factors that Influence Academic Teacher's**

The idea of distance learning as the concept of learning at one's own pace independent of time and place, originated in 19th century. The Open University of United Kingdom, which introduced blended learning in 1969, had the crucial role in introducing the distance learning into the higher education. The term e-learning (electronically supported learning) was introduced in 1995, and its most common definition is: learning and teaching using information and communications technology, or more broadly, e-learning technology. Blended model of learning combines different models of learning and teaching: in traditional classroom (which provides e-learning technologies) and virtual learning environment. Virtual Learning Environment (VLE) is a component or subsystem of the Managed Learning Environment (MLE), where students and teachers take part in different types of online interaction, and whose focus is on managing and facilitating the learning process while providing the required resources. The commonly used synonym for VLE is Learning Management System (LMS). MLE includes various information systems and processes of the higher education, and together with VLE they make a part of virtual

One of the definitions of virtual campus is: "...refers to a specific format of distance education and on-line learning in which students, teaching staff and even university administrative and technical staff mainly 'meet' or communicate through technical links"

Higher education teacher can find the service of using VLE at certain institutions, which can use the service developed within the institution or at the university level. In practice, most commonly used are commercial software packages (integrated set of tools for communication, knowledge evaluation, collaboration, monitoring and other) such as

Introducing e-learning into higher education institution brings about changes on organizational, economical and technical level, however, the practice shows that e-learning has been introduced into such institutions in various ways which resulted in different quantity and quality of the education processes using e-learning technology. To improve the effectiveness of e-learning, the need occurred for developing the quality management system in the field of e-learning (Kermek et al., 2007). Those standards are: ISO/IEC 19796-

WebCT and Blackboard, and among Open Source packages, Moodle and Claroline.

campus (The Joint Information System Committee [JISC], 2011).

(benchmarking of Virtual Campuses [BENVIC], 2011).

**1. Introduction**

**Acceptance of E-Learning Technology in**

**Blended Learning Environment**

*Polytechnic of Rijeka, Business Department, Rijeka,*


**1. Introduction**

Malnutrition is a highly prevalent condition among oncology patients [1]. Up to 70% of cancer patients are at increased risk for malnutrition [2,3], a condition that is strongly associated with higher mortality and morbidity, functional decline, prolonged hospital stays and increased health care costs [4–9]. The pathophysiology of malnutrition in cancer patients is complex and involves different direct and indirect mechanisms, including inflammation, direct tumor effects, chemotherapy-induced effects and a decrease in appetite [10–12]. In addition, a reduced nutrient intake leads to protein and energy deficits, which in turn lead to muscle wasting and impairment of muscle strength [13,14].

Handgrip strength (HGS) measured through dynamometry is an important tool for the assessment of sarcopenia [15]. HGS has been proposed as an easy-to-use, noninvasive,

**Citation:** Tribolet, P.; Kaegi-Braun, N.; Gressies, C.; Baumgartner, A.; Wagner, K.-H.; Stanga, Z.; Schuetz, P. Handgrip Strength Values Depend on Tumor Entity and Predict 180-Day Mortality in Malnourished Cancer Patients. *Nutrients* **2022**, *14*, 2173. https://doi.org/10.3390/nu14102173

Academic Editors: Omorogieva Ojo and Amanda R Amorim Adegboye

Received: 21 April 2022 Accepted: 20 May 2022 Published: 23 May 2022

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

**Copyright:** © 2022 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/).

objective and inexpensive tool to detect and monitor changes in nutritional status, and to predict functional decline during hospitalization and post-discharge [16–19]. HGS correlates with nutritional status and may detect changes in functional capacity in an early stage, before changes in the body composition are manifest [20]. Therefore, the use of HGS has been advocated by different international guidelines as an important adjunct in the assessment of malnutrition [21–24]. Moreover, for the population of cancer patients, studies have suggested that lower HGS is associated with higher risks for mortality and sarcopenia, as well as a decrease in quality of life (Qol) [25].

Based on published data of cancer patients, HGS reference values for this population are expected to be lower compared to healthy people [26–30], but there is a lack of data on reference values for this specific patient population. Herein, we investigated sex-specific HGS levels by tumor entity and additionally studied the prognostic information regarding 180-day all-cause mortality and other adverse outcomes from cancer patients included in the Effect of Early Nutritional Support on Frailty, Functional Outcome, and Recovery of Malnourished Medical Inpatients Trial (EFFORT) [31]. Knowledge of such data may provide health care workers with information about expected results when managing cancer patients in their clinical routine.

#### **2. Material and Methods**

#### *2.1. Study Design and Setting*

This is a secondary analysis of the EFFORT trial, which was a pragmatic, investigatorinitiated, open-label, randomized controlled trial conducted in eight Swiss hospitals between April 2014 and February 2018. The original study investigated the effects of a protocol-guided individualized nutritional treatment algorithm on medical outcomes in patients at nutritional risk. The protocol and the main results [31,32], as well as several predefined secondary analyses [33–42], have been previously published. The EFFORT trial was registered with ClinicalTrials.gov, number NCT02517476.

The Ethics Committee of Northwest/Central Switzerland (EKNZ) approved the study protocol in January 2014 (EKNZ; 2014\_001). The eight participating sites were secondary and tertiary care hospitals in Switzerland and included the University Clinic in Aarau, the University Hospital in Bern, the cantonal hospitals in Solothurn, Lucerne, St. Gallen, Baselland, Muensterlingen and the hospital in Lachen.


**4.1.3 Process shrinkages on the warp and weft directions**

Fabric shrinkages of PET on the warp and weft directions in the each step on the dyeing and finishing processes were calculated using warp and weft fabric densities as equation (1) and (2) shown in bellows.

Warp shrinkage (%)

64 Woven Fabrics

Weaving tensions on 7 kinds of weaving machines were measured using Dafat tension meter which is shown in Fig. 4. Measured position was between tension roller and drop wire on the loom. Various yarn tensions on the each heald frame from 1st to 5th were

Yarn tension along full width of each loom was also measured on the 5th heald frame from left side to right on the back of the loom. The weaving efficiency in each loom was measured

At the upper state of the heald frame, the distance from the fixed guide of heald frame to the upper line of frame is the amount of upper shedding, the lowest state of the heald frame is

The warp movement is calculated by the difference between the amount of upper shedding

**4. Measurement and assessment 4.1 Assessment in the weaving process**

measured at the vicinity of the center of loom.

by number of end breaks both warp and weft per 100,000 picks.

**4.1.1 Warp and weft tensions**

Fig. 4. DEFAT tension meter

Fig. 5. Diagram of shed amount

the amount of lower shedding.

**4.1.2 Measurement of shed amount**

and lower shedding, which is shown in Fig. 5.


**Jiabin Li, Xianwei Liu, Yang Zou, Yichu Deng, Meng Zhang, Miaomiao Yu, Dongjiao Wu, Hao Zheng and Xinliang Zhao**

Factors Affecting COVID-19 Preventive Behaviors among University Students in Beijing, China: An Empirical Study Based on the Extended Theory of Planned Behavior Reprinted from: *Int. J. Environ. Res. Public Health* **2021**, *18*, 7009, doi:10.3390/ijerph18137009 . . . **241**

**Hana Sonbol, Hadil M. Alahdal, Rasis A. Alanazi, Khawla Alsamhary and Fuad Ameen** COVID-19 Pandemic Causing Depression in Different Sociodemographic Groups in Saudi Arabia

Reprinted from: *Int. J. Environ. Res. Public Health* **2021**, *18*, 6955, doi:10.3390/ijerph18136955 . . . **259**


*Article* **Celiac Disease-Type Tissue Transglutaminase Autoantibody Deposits in Kidney Biopsies of Patients with IgA Nephropathy**

**Rakel Nurmi 1, Ilma Korponay-Szabó 1,2,3, Kaija Laurila 1, Heini Huhtala 4, Onni Niemelä 5,6, Jukka Mustonen 6,7, Satu Mäkelä 6,7, Katri Kaukinen 1,7 and Katri Lindfors 1,\***

**Citation:** Nurmi, R.; Korponay-Szabó, I.; Laurila, K.; Huhtala, H.; Niemelä, O.; Mustonen, J.; Mäkelä, S.; Kaukinen, K.; Lindfors, K. Celiac Disease-Type Tissue Transglutaminase Autoantibody Deposits in Kidney Biopsies of Patients with IgA Nephropathy. *Nutrients* **2021**, *13*, 1594. https:// doi.org/10.3390/nu13051594

Academic Editors: Vassilios Liakopoulos and Stefano Guandalini

Received: 26 March 2021 Accepted: 8 May 2021 Published: 11 May 2021

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

**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/).

**Abstract:** An association between celiac disease and IgA nephropathy (IgAN) has been suggested. In celiac disease, in addition to circulating in serum, IgA-class tissue transglutaminase (tTG) autoantibodies are deposited in the small bowel mucosa and extraintestinal organs. In this case series of IgAN patients with or without celiac disease, we studied whether celiac disease-type IgA-tTG deposits occur in kidney biopsies. The study included nine IgAN patients, four of them with celiac disease. At the time of the diagnostic kidney biopsy serum tTG autoantibodies were measured and colocalization of IgA and tTG was investigated in the frozen kidney biopsies. Three IgAN patients with celiac disease had IgA-tTG deposits in the kidney even though in two of these the celiac disease diagnosis had been set years later. These deposits were not found in a patient with already diagnosed celiac disease following a gluten-free diet. Of the five non-celiac IgAN patients, three had IgA-tTG deposits in the kidney. We conclude that tTG-targeted IgA deposits can be found in the kidney biopsies of gluten-consuming IgAN patients but their specificity to celiac disease seems limited.

**Keywords:** IgA nephropathy; celiac disease; tissue transglutaminase autoantibody; tissue transglutaminasetargeted IgA deposits

#### **1. Introduction**

Celiac disease, an immune-mediated enteropathy, is driven by the ingestion of cereals, wheat, rye, and barley containing gluten and characterized by a disease-specific autoantibody response targeting tissue transglutaminase (tTG) [1]. During gluten consumption, IgA-class tTG autoantibodies circulate in serum but are also deposited in the small bowel mucosa, where they are bound to their antigen tTG around mucosal capillaries and on the basement membrane below the mucosal epithelium [2,3]. Interestingly, these small intestinal IgA deposits may be present even before the development of small bowel mucosal villous atrophy or the detection of the tTG autoantibodies in the circulation, and they may predict forthcoming manifest celiac disease [3–5]. Moreover, IgA deposits in the gut have also been found in celiac disease patients with negative serum tTG autoantibodies [6,7]. Upon introduction of a gluten-free diet (GFD), the gold standard treatment for celiac disease, these small intestinal mucosal deposits disappear along with serum tTG autoantibodies [4,8]. IgA-class tTG-targeted autoantibody deposits can be also found in

several other tissues, including liver, muscle, and brain, often coinciding with extraintestinal manifestations of celiac disease affecting the organ in question (e.g., hepatitis, muscle weakness, and ataxia) [3,7]. However, little is known about the occurrence of such extraintestinal IgA-tTG deposits long before diagnosis of celiac disease or their dependence on the presence of gluten in the diet.

It has been proposed that celiac disease may be associated with IgA nephropathy (IgAN), globally the most common primary chronic glomerular disease [9,10]. The diagnostic hallmark of IgAN is the predominance of hypo-galactosylated IgA1 deposits in the mesangium of the glomeruli [11]. In IgAN, the interaction between environmental antigens, dysregulation of IgA immune responses and pathogenic circulating IgA complexes eventually leads to IgA1 deposits in the kidney [12]. Although celiac disease and IgAN target different organs, they have a great deal in common. As mentioned above, aberrant IgA response is involved in both diseases [13]. tTG is known to play a decisive role in the pathogenesis of celiac disease by modifying wheat gluten-derived gliadin into a more immunogenic form [14]. Although the role of tTG in IgAN is less clear, studies conducted in both human patients and mice suggest that tTG is needed for the development of IgANtype mesangial IgA deposits and the impairment of the clinical course of IgAN [15,16]. The ingestion of dietary gluten is required for the development of celiac disease, and may also promote the development of IgAN, at least in mice [14,17,18]. In addition, GFD being the only treatment for celiac disease, it is intriguing that some reports have described clinical improvement of IgAN with the same diet [19–21].

The association between celiac disease and IgAN is still being actively researched. In this case series we investigated whether celiac disease-type tTG-targeted IgA autoantibody deposits occur in the kidney biopsies of IgAN patients with or without concomitant or subsequent celiac disease.

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

#### *2.1. Patients and Clinical Data*

This retrospective study reports a case series of nine adult IgAN patients on whom diagnostic kidney biopsy was performed during the period 1981–1987 at Tampere University Hospital, Finland. IgAN was defined as glomerulonephritis with typical light microscopy features and IgA as the sole or main glomerular immunofluorescence finding [9]. Four of the IgAN patients also had diagnosed celiac disease. Five non-celiac patients who had received their IgAN diagnoses during the same period were selected as controls. The patients were followed-up until recent available laboratory results or death. Clinical data at the time of the IgAN diagnoses and at follow-up were collected from the medical records and included demographic data, creatinine values, and data on celiac disease diagnosis. The outcomes (chronic dialysis, kidney transplant, and death) were documented. Creatinine values and the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation were used to determine estimated glomerular filtration rate (eGFR). eGFR ≥90 indicates normal kidney function, eGFR 30–59 moderate renal impairment, eGFR 15–29 severe renal impairment, and eGFR <15 renal failure [22].

#### *2.2. Determination of Serum and Tissue tTG-Targeted IgA Autoantibodies*

Stored frozen (−80 ◦C) serum samples taken at the time of kidney biopsy were used to identify IgA-class tTG autoantibodies using the ELIA Celikey assay (Celikey®, Phadia, GmbH, Freiburg, Germany) according to manufacturers' instructions. Values higher than 7.0 U/mL were regarded as positive.

Celiac disease-type tissue deposits with colocalization of IgA and tTG were determined in frozen biopsies by evaluators blinded to the clinical data. These included kidney biopsies for diagnosing IgAN and small bowel mucosal biopsies of two celiac disease patients taken at the time of the celiac disease diagnoses. The deposits were detected using the technique described earlier by Korponay-Szabó et al. [3] from snap frozen biopsies embedded in optimal cutting temperature compound (OCT, Tissue Tek, Sakura Finetek

Europe B.V., AJ Alphen aan den Rijn, The Netherlands). The frozen sections of 5 μm thickness were stained with fluorescein isothiocyanate (FITC)-labeled rabbit antibodies against human IgA (Dako AS, Glostrup, Denmark) at a dilution of 1:40 in phosphate buffered saline and with monoclonal mouse antibodies against tTG (CUB7402, NeoMarkers, Fremont, CA, USA), which were detected with rhodamine-conjugated anti-mouse immunoglobulin rabbit antibodies (Dako) diluted 1:200 in phosphate buffered saline.

The kidney samples were investigated for the presence of tTG and IgA. IgA deposits around the basement membrane of extraglomerular blood vessels, of the parietal layer of Bowman's capsules and of the proximal or distal tubuli in colocalization with tTG were regarded as celiac disease-type deposits. Although the glomerular capillaries also contain tTG around their basement membrane, IgA deposition on tTG within the glomeruli was not clearly discernible because all patients had extensive mesangial IgA deposits related to IgAN itself. Therefore, glomerular IgA was not taken into account for celiac disease-type deposit evaluation.

In the small bowel mucosal biopsies, subepithelial IgA deposits found on the basement membrane below the villous and crypt epithelium and around the mucosal blood vessels were regarded as celiac disease-type IgA deposits, as in non-celiac subjects small-bowel mucosa IgA is detected only inside the plasma and epithelial cells [3,6]. Colocalization of IgA and tTG was regarded as celiac disease-type deposits.

#### *2.3. Ethical Considerations*

Informed consent for the study was obtained from the patients. The research protocol (E99105, R20056) was approved by the Regional Ethics Committee of Pirkanmaa Hospital District. The study protocol follows the ethical guidelines of the Declaration of Helsinki.

#### **3. Results**

By definition, all nine IgAN patients in our case series had glomerular IgA deposits characteristic of IgAN (Figure 1a). Four of the subjects were female (Table 1). At the time of the kidney biopsy and IgAN diagnosis their median age was 34 years (range 20–50 years) and eight of them were on a normal gluten-containing diet (Table 1). One patient (9M) with previously diagnosed celiac disease was on GFD. One patient (1F) was diagnosed with celiac disease during the same treatment episode as the diagnosis of IgAN. During follow-up two additional subjects (2M and 3M) received celiac disease diagnoses after eight and ten years respectively. At the time of celiac disease diagnoses, both of these cases had tTG-targeted autoantibodies in serum and IgA-tTG deposits in the small bowel mucosal biopsies (Figure 2).

In the kidney biopsies, celiac disease-type deposits characterized by colocalization of IgA and tTG were detected in all three patients with both IgAN and celiac disease on a normal gluten-containing diet (Figure 1b–d, Table 1). It is noteworthy that in two patients (2M and 3M) diagnosed with celiac disease during follow-up, the celiac diseasetype IgA-tTG autoantibody deposits were already present in the renal tissue at the time of the kidney biopsies taken eight and ten years prior to celiac disease diagnosis. In all three patients with both IgAN and celiac disease, connective tissue IgA deposits were found around both proximal and distal tubuli. Moreover, in two of these patients (1F and 3M) deposits were also seen in the periglomerular region around Bowman's capsule. Interestingly, tTG autoantibody levels in the serum taken at the time of kidney biopsy and determined retrospectively from stored samples were already elevated in all these celiac disease patients, including those subsequently diagnosed with celiac disease during follow-up (Table 1).

Of the five IgAN patients without celiac disease, three had celiac disease-type IgA-tTG deposits around the proximal and distal tubuli in the kidney (4F, 5F, 6M) without having elevated levels of serum tTG autoantibodies at the cross-sectional serologic evaluation at diagnosis (Table 1). Of the remaining two IgAN patients without celiac disease (7F, 8M), neither had celiac disease-type IgA-tTG deposits in the kidney. Of these patients, 7F had no

serum tTG autoantibodies, while 8M had no serum sample available for analysis. Patient 9M on GFD due to earlier diagnosed celiac disease, had no celiac disease-type tTG-targeted IgA deposits in the diagnostic kidney biopsy, although serum tTG autoantibody levels were still slightly elevated.

**Figure 1.** Immunofluorescent staining for immunoglobulin A (IgA) (green) and tissue transglutaminase (tTG) (red), and their colocalization (yellow) in the renal biopsies taken at the time of the IgA nephropathy (IgAN) diagnosis. (**a**) In a patient with IgAN without celiac disease, the IgA is only found in the glomerular mesangium. No colocalization of IgA and tTG is detected; (**b**) representative figure demonstrating colocalization of IgA and tTG (yellow) in the extracellular matrix around the renal tubuli in patients with both IgAN and celiac disease; (**c**) IgA; (**d**) tTG staining in the same specimen. Magnification 20x.

**Table 1.** Background and follow-up data and biopsy findings among nine patients with IgA nephropathy (IgAN).

IgAN, IgA nephropathy; CD, celiac disease; tTG, tissue transglutaminase; eGFR, estimated glomerular filtration rate. tTG autoantibody levels higher than 7.0 U/mL were regarded as positive. eGFR ≥ 90 indicates normal kidney function, eGFR 60–89 mild loss of kidney function, eGFR 30–59 moderate renal impairment, eGFR 15–29 severe renal impairment and eGFR < 15 renal failure. 1 Started from kidney biopsy. 2 Risk-group screening. 3 The diagnosis of CD was made before the diagnosis of IgAN and the patient followed gluten-free diet at the time of kidney biopsy. Initial serum tTG autoantibody level was not known. 4 Exact time of diagnosis of CD was not known.

> The median follow-up for all patients was 28 (range 5–35) years (mean 23, standard deviation 11 for comparison). Four patients with IgAN and celiac disease were followed-up for a median 28 years (range 5–35) (mean 24, standard deviation 13 for comparison). Two of these had died and two suffered from severe loss of kidney function (eGFR 17 and 23 respectively). Five IgAN patients without celiac disease were followed-up for a median 20 years (range 10–35) (mean 22, standard deviation 11 for comparison). One had normal kidney function, two mild loss of kidney function, and two moderate renal impairment (Table 1). None of these patients required dialysis treatment. No follow-up data on serum tTG autoantibody levels were available.

**Figure 2.** Small bowel mucosal biopsy of a celiac disease patient at the time of diagnosis of celiac disease stained for celiac disease-type IgA-tTG deposits. (**a**) Immunofluorescent staining demonstrating the colocalization of IgA and tTG (yellow); (**b**) staining for IgA (green), and (**c**) tissue transglutaminase (tTG) (red) in the same specimen. Magnification 20x.

#### **4. Discussion**

In this case series, we found that, during ingestion of gluten, celiac disease-type tTGtargeted IgA deposits were present in the kidney biopsies of all patients with both IgAN and celiac disease and thus our results are in line with the case report by Costa et al. [23]. Two of the IgAN patients with celiac disease (2M, 3M) were found to have IgA-tTG deposits in the kidney years before the diagnosis of celiac disease. Parallel deposits were also detected in the small bowel mucosal biopsies of these two patients (2M, 3M) at the time of celiac disease diagnosis, but unfortunately no small bowel mucosal biopsies taken at the time of IgAN diagnosis were available for the determination of IgA-tTG deposits. However, as small bowel mucosal IgA-tTG deposits may be present prior to small bowel mucosal damage diagnostic for celiac disease [4], the finding of celiac disease-type IgA-tTG deposits in the kidney is interesting and suggests that such deposits may also occur prior to celiac disease diagnosis. It must be noted, however, that both patients (2M, 3M) with IgA-tTG deposits in the kidney also had tTG autoantibodies in serum at the time of IgAN diagnosis. This would suggest that they already had celiac disease at this point, even though the clinical diagnosis of celiac disease came only later. In fact, one of these patients (3M) had tTG autoantibody levels 10 times above the cut-off and this would have been sufficient to warrant a diagnosis of celiac disease under the current guidelines [24]. In any case, as patient 9M with diagnosed celiac disease and already on a GFD at the time of kidney biopsy did not have celiac disease-type IgA-tTG deposits in the kidney, this would suggest that these deposits may be gluten-dependent. The fact that this patient had circulating tTG autoantibodies in low concentrations casts doubt on the strictness of the diet in this patient but may also suggest that these celiac disease-type deposits may disappear from the extraintestinal organs even before the complete clearance of autoantibodies from circulation.

In our study the IgA-tTG deposits in the kidney were not specific to celiac disease patients since they were also found in three non-celiac IgAN patients without serum tTG autoantibodies. Although no data is available on the presence of extraintestinal IgAtTG deposits in seronegative celiac disease, such patients have been reported to have such deposits in the small bowel mucosa [6]. Unfortunately, no small bowel mucosal specimens taken at the time of IgAN diagnosis were available for determination of mucosal morphology and IgA-tTG deposits, and therefore we cannot be certain whether these IgAN patients with IgA-tTG deposits in the kidney were indeed antibody-negative celiac disease patients. Moreover, serum samples or small bowel mucosal biopsies during follow-up were not collected and thus it cannot be ascertained whether these individuals developed

celiac disease later. However, it is interesting that our earlier studies describe small bowel inflammation and stress in IgAN in the absence of celiac disease [25,26]. Furthermore, increased immune reactivity to dietary antigens, including gluten, has been suggested among IgAN patients even without overt dietary intolerance [20,27]. Intestinal IgA-tTG deposits have been reported in patients without celiac disease and also in a patient with gluten ataxia, in whom the deposits were also found in the brain [7,28]. Hence the renal IgAtTG deposits in IgAN patients without celiac disease could indicate a similar phenomenon. It is possible that the colocalization of IgA-tTG deposits in the kidney among IgAN patients with celiac disease is a familiar antigen-antibody interaction, while that among patients with IgAN reflects another type of molecular phenomenon related, for instance, to increased expression of tTG in the renal biopsies of IgAN patients [16].

In our study the patients with IgAN and celiac disease seemed to have worse outcomes of IgAN than did the patients with IgAN only. This finding is interesting in the light of our earlier study and the Swedish study by Rehnberg and co-workers showing that prognosis in IgAN may be poorer with concomitant comorbid bowel disease [29,30]. However, given the small number of patients and the fact that several factors affected the outcome of IgAN patients [31], conclusions concerning the renal survival of IgAN patients with celiac disease cannot be drawn on the basis of our data. Additionally, the follow-up of IgAN patients with celiac disease was longer, which may likewise affect this finding. In any case, the impact of the celiac disease-type IgA-tTG deposits in the kidney seemed not to be related to renal function or outcome, as also suggested by earlier findings of IgA in the kidneys of celiac disease patients without any renal problems [32].

To conclude, tTG-targeted IgA deposits were found in the kidney biopsies of glutenconsuming IgAN patients with or without known celiac disease. The significance of this interesting finding remains open and therefore in the future larger studies, preferably with more data on small bowel histochemistry and regular follow-up serology for tTG antibodies, are needed to investigate the association between celiac disease and IgAN in greater detail.

**Author Contributions:** Conceptualization and methodology, R.N., I.K.-S., J.M., S.M., K.K. and K.L. (Katri Lindfors); formal analysis and investigation, R.N., I.K.-S., K.L.(Kaija Laurila), H.H., O.N., K.K. and K.L. (Katri Lindfors); writing—original draft preparation, R.N. and K.L. (Katri Lindfors); writing—review and editing, R.N., I.K.-S., K.L. (Kaija Laurila), H.H., O.N., J.M., S.M., K.K. and K.L. (Katri Lindfors); supervision, J.M., S.M., K.K. and K.L. (Katri Lindfors); funding acquisition, R.N., K.K. and K.L. (Katri Lindfors). All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital, the Academy of Finland, the Sigrid Jusélius Foundation, the Finnish Celiac Society, Tampere University Doctoral School, the Finnish Kidney and Liver Association, research grants NKFI 120392 from the Hungarian National Research, Development and Innovation Fund and the GINOP-2.3.2-15-2016-00015 and Interreg Danube DTP571 CD SKILLS projects, co-financed by the European Union and the Hungarian State.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Pirkanmaa Hospital District (protocol codes E99105, R20056).

**Informed Consent Statement:** Informed consent was obtained from the subjects involved in the study or the Ethics Committee of Pirkanmaa Hospital District in cases of deceased patients.

**Data Availability Statement:** The data are not publicly available due to ethical and privacy reasons.

**Acknowledgments:** The valuable work of Ole Wirta (MD, PhD), Suvi Kalliokoski (PhD), Immo Rantala (PhD), and Johannes Valkonen is greatly appreciated.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**