ISSN 0030- 6096

O S A K A
C I T Y

Vol.

69

No.2

M E D I C A L

2023

J O U R N A L

V o l . 6 9 ,N o . 22 0 2 3

PUBLISHED BYOSAKA CITY MEDICAL ASSOCIATIONOSAKA JAPANOsaka City Medical Journal
Vo l . 6 9 , N o . 2 ,December 2023

CONTENTS
Analysis of Anti-laminin γ1 Autoantibodies in Anti-p200 Pemphigoid by

page

Immunoblotting Using Cultured Cells
DAISUKE HAYASHI, TAKASHI HASHIMOTO, MAKO MINE,NORITO ISHII, KENTARO IZUMI, and DAISUKE TSURUTA ･･････････････････････････････････････ 51
Prolonged Olfactory Dysfunction in the COVID-19 Era; Etiological
Analysis in a Single-centered Cohort
HIROKO KAWAI, KOUSUKE HASHIMOTO, YUICHI TERANISHI,YU NAKAGAMA, YASUTOSHI KIDO, and KISHIKO SUNAMI ･････････････････････････････････････ 61
Outcomes of COVID-19 Pneumonia Patients Complicated with Barotrauma
under Mechanical Ventilation
MASAHIRO MIYASHITA, RYO DEGUCHI, SEIYA KURIMASA,FUMIAKI WAKITA, HOSHI HIMURA, AKIHIRO KAWAMOTO,KENICHIRO UCHIDA, TETSURO NISHIMURA, HIROMASA YAMAMOTO,
and YASUMITSU MIZOBATA ････････････････････････････････････････････････････････････････････････････ 67― Case Report ―A Case of Conversion Surgery for Pancreatic Head Cancer with Liver Metastases
and Renal Anemia after Combination Therapy with Anticancer Agentsand a Hypoxia-inducible Factor Prolyl Hydroxylase Inhibitor
GO OHIRA, RYOSUKE AMANO, SHOGO TANAKA, KENJIRO KIMURA,HIROJI SHINKAWA, KOHEI NISHIO, MASAHIKO KINOSHITA,
JUN TAUCHI, DAISUKE SHIRAI, TAKUMA OKADA, TAKAHITO KAWAGUCHI,NAOKI TANI, and TAKEAKI ISHIZAWA ･･･････････････････････････････････････････････････････････････ 75
Domino Reconstruction of a Mycotic Aneurysm in the Common Iliac Artery
Using an Autologous Graft from the Contralateral Iliac Artery
AKIHIRO SUMIYA, YASUYUKI BITO, TAKANOBU AOYAMA,MASANORI SAKAGUCHI, YURIKO KIRIYA,
and TAKASHI MURAKAMI ･･･････････････････････････････････････････････････････････････････････････････ 83

Osaka City Med. J. Vol. 69, 51-59, 2023

Analysis of Anti-laminin γ1 Autoantibodies in Anti-p200
Pemphigoid by Immunoblotting Using Cultured Cells
DAISUKE HAYASHI1), TAKASHI HASHIMOTO2), MAKO MINE2), NORITO ISHII3),
KENTARO IZUMI4), and DAISUKE TSURUTA2)
Department of Dermatology1), Graduate School of Medicine, Osaka City University;
Department of Dermatology2), Graduate School of Medicine, Osaka Metropolitan University;
Department of Dermatology3), Kurume University School of Medicine; andDepartment of Dermatology4), Hokkaido University Graduate School of Medicine

Abstract
BackgroundAnti-p200 pemphigoid is a subepidermal autoimmune bullous disease involving IgG autoantibodies
to the 200-kDa antigen. Although previous studies suggested that patient sera react with the 200kDa laminin γ1, it is possible that another 200-kDa autoantigen is present. Here, we examined
whether the anti-p200 pemphigoid patient sera react exclusively with laminin γ1 by performing
experiments using LAMC1-knock-out (KO) cells produced by the use of CRISPR-Cas9.
Methods
We performed immunoblotting experiments using normal human dermal extract and extracts ofvarious cultured cells, including LAMC1-KO HaCaT cells.
Results
While all the anti-p200 pemphigoid patient sera and the anti-laminin γ1 monoclonal antibody(mAb) reacted with the 200-kDa protein in normal human dermal extract, the mAb detected a smaller
190-kDa laminin γ1 molecule in extracts of various cultured cells. However, none of the patient sera
reacted with the 190-kDa laminin γ1 in extracts of either intact or LAMC1-KO HaCaT cells.
Conclusions
We could not identify the 200-kDa antigen as laminin γ1 because the patient sera did not reactwith laminin γ1 in HaCaT cells. The reason may be that the 190-kDa laminin γ1 produced by thecultured cells lacks post-translational modifications necessary for autoantibody binding. However,this novel approach using the CRISPR-Cas9 technique should be useful for the identification of true
autoantigens in patient sera that react with laminin γ1 in cultured cells.
Key Words: ‌Anti-p200 pemphigoid; CRISPR-Cas9; HaCaT cell; Immunoblotting;
Laminin γ1.

Received January 19, 2023; accepted March 3, 2023.
Correspondence to: Takashi Hashimoto, MD.
Department of Dermatology, Graduate School of Medicine, Osaka Metropolitan University,
1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
Tel: +81-6-6646-6630; Fax: +81-6-6646-6630
E-mail: hashyt@gmail.com- 51 -

Hayashi et al

Introduction
Anti-p200 pemphigoid was first described in 1996 by Chen et al1) and Zillikens et al2) as a novel
entity of subepidermal autoimmune bullous disease with IgG autoantibodies against an unknown
200-kDa antigen. Anti-p200 pemphigoid clinically shows blisters, erosions, and urticaria-likeerythema, with a tendency to the development of smaller vesicles 3-7). Mucosal lesions occasionallydevelop. The association with psoriasis has been reported in Japan and Asian countries, but not in
European countries 3-7). Histopathology shows subepidermal blisters and the inf lammatory cell
infiltrate of neutrophils and eosinophils in the upper dermis. Direct immunof luorescence (IF) shows
linear IgG and C3 deposits at the basement membrane zone (BMZ). Indirect IF of normal humanskin detects IgG anti-BMZ antibodies, which react with the dermal side of 1 M NaCl-split normal
human skin. Immunoblotting of normal human dermal extracts detects a 200-kDa autoantigen.
In 2009, Dainichi et al identified the 200-kDa protein as laminin γ1 by proteomics 8). In
immunoblotting of normal human dermal extracts, they found that patient sera and an anti-laminin
γ1 monoclonal antibody (mAb) reacted with the identical 200-kDa protein band. Furthermore, 90% of
32 patient sera reacted with recombinant laminin γ1 protein8). Another mAb, against the C-terminusof laminin γ1, competitively inhibited the reaction of patient sera with the p200 band in immunoblotsof normal human dermal extract. Conversely, the IgG fraction purified from patient sera
competitively inhibited the reactivity of the anti-laminin γ1 mAb in immunoblots of normal humandermal extract. From these results, Dainichi et al concluded that p200 is laminin γ1, and proposed
the name anti-laminin γ pemphigoid for this disease8).
Laminin γ1 is an N-linked glycoprotein with a molecular weight of 200 kDa9-11). In the skin, it
forms heterotrimers such as laminins 311, 321, and 511, which are involved in epidermal and dermalbinding outside the hemidesmosome. Shimanovich et al reported that anti-p200 pemphigoid sera
react with an acidic noncollagenous N-linked glycoprotein of the cutaneous basement membrane 12),which suggests that the 200-kDa autoantigen may be one of the laminin γ subunits.
However, although laminin γ1 is also expressed in various tissues other than skin and mucosa8),
anti-p200 pemphigoid patients do not develop any lesions in other organs. Therefore, it is unclear
why the autoantibodies in the patient sera react selectively with laminin γ1 in the skin. Dainichi etal speculated that the sera of anti-p200 pemphigoid patients might recognize laminin γ1 with skinspecific post-translational modifications and thus induce only the skin lesions8). However, the few
attempts to unravel the pathogenic activity of anti-laminin γ1 antibodies have so far been
unsuccessful13-15). Thus, it is still conceivable that, other than laminin γ1, there is an unknown 200kDa autoantigen that might be the true pathogenic autoantigen.
Here, we examined whether the anti-p200 pemphigoid patient sera react truly and exclusively
with laminin γ1. For this purpose, we generated laminin γ1-knock-out (KO) HaCaT cells andexamined the reactivity of anti-p200 pemphigoid patient sera by immunoblotting of extracts of boththe intact HaCaT cells and the LAMC1-KO HaCaT cells.

Methods
This study was approved by Ethics Committees of Osaka Metropolitan University (No. 2019-055),
and adhered to Declaration of Helsinki principles.
Patient sera and normal control sera
We used 15 sera from anti-p200 pemphigoid patients; they were stored at the Department of- 52 -IB Study of Laminin γ1 for Anti-p200 Pemphigoid

Dermatology, Osaka Metropolitan University, and the Department of Dermatology, Kurume University.
All the patients fulfilled the following diagnostic criteria: linear deposits of IgG and/or C3 in the BMZ
by direct IF, the presence of IgG anti-BMZ antibodies by indirect IF using normal human skin,positive IgG reactivity to the dermal side of 1 M NaCl-split normal human skin by indirect IF, andthe detection of the 200-kDa antigen in immunoblots of normal human dermal extract. The sera werestored at −80℃ for the long term. When used for experiments, the sera were thawed and then stored
again at −20℃ for further use. Normal sera from four volunteers were used as negative controls.
Production of a laminin γ 1-KO HaCaT cell line by the use of CRISPR-Cas9
To establish a Cas9-expressing HaCaT cell line, HaCaT cells were infected with pLenti-EF1aCas9-Puro lentiviral particles (Applied Biological Materials Inc., Richmond, BC, Canada) according to
the manufacturerʼs instructions. Using Lipofectamin RNAi Max (Thermo Fisher Scientific, Waltham,
MA, USA), the cloned Cas9-expressing HaCaT cells were transfected with control gRNA (Integrated
DNA Technologies, Coralville, IA, USA, Alt-R CRISPR-Cas9 Negative Control crRNA, 1072254) orgRNA targeting laminin γ1 (sequence: CTTAATCGCCTGAACACTTT) (Integrated DNA Technologies,
Coralville, IA, USA), coupled to tracRNA (Integrated DNA Technologies, Coralville, IA, USA,1075927). Subsequently, a LAMC1-KO HaCaT cell line was cloned.
Cell cultures
HaCaT cells (CLI Cell Lines Service, Eppelheim, Germany, 300493), DJM-1 cells, and SCC cells
were cultured in Dulbeccoʼs modified Eagleʼs medium (DMEM) (Fujifilm Wako Pure Chemical
Corporation, Osaka, Japan, 04429765) supplemented with penicillin-streptomycin solution (Fujifilm
Wako Pure Chemical Corporation, Osaka, Japan, 168-23191) and 5% filter-sterilized fetal bovine
serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA, 10270106). Normal human epidermal
keratinocytes (NHEKs) (Promo cell, Heidelberg, Germany, C12003) were cultured using keratinocyte
growth medium (Promo cell, Heidelberg, Germany, C-20011). Normal human neonatal dermal
fibroblasts (ATCC, Manassas, VA, USA, PCS-201-010) were cultured in fibroblast basal medium(ATCC, Manassas, VA, USA, PCS-201-030) supplemented with fibroblast growth kit-lower serum(ATCC, Manassas, VA, USA, PCS-201-041). The cell extracts were prepared by scraping the cells at
80%-90% conf luency in SDS sample buffer for electrophoresis.
Indirect immunofluorescence analyses
Indirect immunof luorescence analyses of normal human skin and 1 M NaCl-split normal humanskin were performed as previously described3,8).
Immunoblot analysis
Running buffer for electrophoresis was prepared by adding MOPS running buffer 20×(Thermo
Fisher Scientific, Waltham, MA, USA, NP0001) and 0.25% of antioxidant (Thermo Fisher Scientific,
Waltham, MA, USA, NP0005) to distilled water. Next, 10 μL samples were applied to the lanes of a
3%-8% Tris-acetate gel (Thermo Fisher Scientific, Waltham, MA, USA, EA0375BOX) and were
electrophoresed at 200 V for 1 h. The proteins in the gel were transferred to PVDF membrane (BioRad Laboratories, Inc. Hercules, CA, USA, cat #1704156). The membranes were then blocked with 3%
skim milk in PBS for 1 h, and washed with PBS with 0.5% Tween-20 in PBS (PBS-T) for 10 min, 3 times.
As primary antibodies, patient sera were diluted 1:5 in 3% skim milk in PBS, and the anti-laminin
γ1 mouse IgG monoclonal antibody (mAb) (Atlas Antibodies, Stockholm, Sweden, AMAb91137) wasused at 1:1000-1:10000 dilutions in 3% skim milk in PBS. Primary antibodies were incubatedovernight at 4℃ and the membranes were then washed for 10 min 3 times in PBS-T. The blots were
- 53 -

Hayashi et althen incubated with secondary antibodies, HRP-anti-human IgG antibody (Agilent Technologies
Dako, Glostrup, Denmark, P0214) at 1:1000 dilution and HRP-anti-mouse IgG antibody(SouthernBiotech, Birmingham, AL, USA, 1031-05) at 1:5000 dilution, for 1 h at room temperature,and then washed for 10 min 3 times in PBS-T. Positive reactivity was detected with an Image Quant
LAS 4000 (GE Healthcare, Chicago, IL, USA) using Super Signal (Thermo Fisher Scientific, Waltham,
MA, USA). We used of the same patientsʼ sera as representative in all the experiments.

Results
Indirect immunofluorescence analyses of anti-p200 pemphigoid patient sera
By indirect immunof luorescence of normal human skin, all 15 anti-p200 pemphigoid patient sera
showed anti-BMZ antibodies that reacted with the dermal side of split skin (Fig. 1, the results of 3
representative sera are shown).

Figure 1. Indirect immunof luorescence of 1 M NaCl-split normal human skin using 3 representative anti-p200
pemphigoid sera (patients1-3). White arrows indicate the reactivity with the dermal side of the split.

Figure 2. Immunoblotting of normal human dermal extract using 3 representative anti-p200 pemphigoid sera. Antilaminin γ1 mAb reacted with the 200-kDa laminin γ1 (lane 1). The same 200-kDa protein also reacted with the 3
representative anti-p200 pemphigoid patient sera (patients 1-3), but not with normal control serum (lane 5). The 238and 171-kDa bands of the molecular weight (MW) markers (lane 6) are indicated by arrows on the right.

- 54 -IB Study of Laminin γ1 for Anti-p200 Pemphigoid

Figure 3. Immunoblotting of extracts of various cultured cells with the anti-laminin γ1 mAb. While this mAb reactedwith the 200-kDa laminin γ1 in normal human dermal extracts (lane 1), it reacted with the 190-kDa laminin γ1 in all
the cultured cells, including HaCaT cells (lane 2), DJM-1 cells (lane 3), SCC cells (lane 4), normal human epidermal
keratinocytes (NHEKs) (lane 5), and dermal fibroblasts (lane 6). The 238- and 171-kDa bands of the molecular weight(MW) markers (lane 7) are indicated by arrows on the right.

Figure 4. Immunoblotting of extracts of intact HaCaT cells and LAMC1-KO HaCaT cells with anti-laminin γ1 mAb.
The mAb reacted with the 190-kDa laminin γ1 in intact HaCaT cell extract (lane 1), and did not detect it in LAMC1KO HaCaT cell lysate (lane 2).

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Hayashi et al

Figure 5. Immunoblotting of extracts of intact HaCaT cells with anti-p200 pemphigoid patient sera. While the antilaminin γ1 mAb detected the 190-kDa laminin γ1 (lane 1), this band was not detected by any of the 3 representative
anti-p200 pemphigoid patient sera (lane 2-4) or by normal serum (lane 5). The 238- and 171-kDa bands of themolecular weight (MW) markers are indicated by arrows on the right (lane 6).

Figure 6. Immunoblotting of extracts of intact HaCaT cells and LAMC1-KO HaCaT cells with anti-p200 pemphigoid
patient sera. The anti-laminin γ1 mAb reacted with the 190-kDa laminin γ1 in intact HaCaT cell extract (lane 1), but
was not detected by any of the 3 representative anti-p200 pemphigoid patient sera in extracts of either intact HaCaT
cells or LAMC1-KO HaCaT cells (lanes 2-7). The 250- and 180-kDa bands of the molecular weight (MW) markers areindicated by arrows on the right (lane 8).

- 56 -IB Study of Laminin γ1 for Anti-p200 Pemphigoid

Immunoblotting of normal human dermal extract with anti-p200 pemphigoid patient sera
In immunoblots of normal human dermal extract, all 15 anti-p200 pemphigoid patient sera
reacted with the 200-kDa protein band (Fig. 2, the results of 3 representative sera are shown), which
was also strongly detected by the anti-laminin γ1 mAb.
Immunoblotting of extracts of various cultured cells by anti-laminin γ 1 mAb
To confirm their expression of laminin γ1, we performed immunoblotting with extracts of various
cultured cells, including HaCaT cells, DJM1 cells, SCC cells, NHEKs, and dermal fibroblasts, using
anti-laminin γ1 mAb. While the anti-laminin γ1 mAb detected the 200-kDa laminin γ1 in normal
human dermal extract (Fig. 3, lane 1), the mAb reacted with an approximately 190-kDa smaller
laminin γ1 in the extracts of all the other cultured cells (Fig. 3, lanes 2-6).
Immunoblotting of intact HaCaT cell extract and LAMC1-KO HaCaT cell extracts with antilaminin γ 1 mAb
To confirm the lack of p200 detection in LAMC1-KO HaCaT cells, we compared the laminin γ1
reactivity of intact HaCaT cells and LAMC1-KO HaCaT cells by immunoblotting. The anti-laminin
γ1 mAb detected the 190-kDa laminin γ1 in immunoblots of intact HaCaT cells, but did not detect it
in LAMC1-KO HaCaT cell extract (Fig. 4), confirming that laminin γ1 was knocked out in the
LAMC1-KO HaCaT cells.
Immunoblotting of intact HaCaT cell extract and LAMC1-KO HaCaT cell extracts with
anti-p200 pemphigoid patient sera
Next, we performed immunoblotting of intact HaCaT cell extract with all 15 anti-p200 pemphigoid
patient sera. However, although the anti-laminin γ1 mAb detected the 190-kDa laminin γ1 in
immunoblots of extracts of intact HaCaT cells, none of the patient sera detected the 190-kDa proteinband in intact HaCaT cell extract (Fig. 5, the results of 3 representative sera are shown). In further
immunoblotting of extracts of both intact HaCaT cells and LAMC1-KO HaCaT cells, the 190-kDa
protein band was not detected in either extract by any of the 15 anti-p200 pemphigoid patient sera
(Fig. 6, the results of 3 representative sera are shown).

Discussion
The aim of this study was to examine whether the anti-p200 pemphigoid sera really andexclusively react with laminin γ1.
First, we confirmed the reactivity of all the 15 anti-p200 pemphigoid sera with the 200-kDa p200
by indirect immunof luorescence of 1 M NaCl-split skin and immunoblotting of normal human dermal
extract. The results confirmed the results of the previous studies by Li et al11).
The first important result in this study was that, by immunoblotting analysis of extracts ofvarious cultured cells using anti-laminin γ1 mAb, all the cultured cells, including HaCaT cells, DJM1
cells, SCC cells, NHEKs, and dermal fibroblasts, expressed laminin γ1 molecules of approximately
190 kDa, which were smaller than the 200-kDa laminin γ1 in human dermal extract.
In the next experiment, we successfully produced a LAMC1-KO HaCaT cell line, using CRISPRCas9. Its lack of laminin γ1 expression was confirmed by immunoblotting with the anti-laminin γ1
mAb.
Next, we examined the 15 patient sera by immunoblotting extracts of both the intact HaCaT cells
and the LAMC1-KO HaCaT cells. However, none of the patient sera showed positive reactivity withthe 190-kDa laminin γ1 in either extract. Thus, we could not confirm that all the patient sera really- 57 -

Hayashi et al

reacted with laminin γ1 with this approach.
The reason for the negative reactivity of patient sera with the smaller 190-kDa laminin γ1 in
HaCaT cells is currently unknown. However, it is conceivable that the cultured cells could not
produce enough post-translational modifications with which the anti-laminin γ1 autoantibodies in
the patient sera react. This speculation is supported by previous reports that N-linked glycosylationof laminin γ1 inf luences its recognition by anti-p200 pemphigoid autoantibodies11,12).
The present results also suggest that other target proteins may be present.

Conclusions
In this study, we could not identify the 200-kDa antigen as laminin γ1 because the patient sera
did not react with laminin γ1 in either intact or LAMC1-KO HaCaT cells. However, this approach
using the CRISPR-Cas9 technique should be useful in identifying true autoantigens in patient sera
that react with laminin γ1 in cultured cells. Future studies to clarify the nature of the pathogenic
autoantibodies in anti-p200 pemphigoid, and particularly to attempt to identify any different
autoantigen, are warranted.
Acknowledgements
All authors have no COI to declare regarding the present study.
We thank Ms. Nami Ikeshita, Department of Dermatology, Hokkaido University Graduate Schoolof Medicine, for her technical assistance. We are grateful to Ms. Noriko Okabayashi and Ms. Yoko
Mukaoku, Graduate School of Medicine, Osaka Metropolitan University, for their secretarial work.
We thank all the patients for their participation. We are grateful to Dr. Xiaoguang Li and Dr. Hua
Qian, Department of Laboratory Medicine, Medical College, Dalian University, Dalian, China, for
critically reading and commenting on the manuscript.
Grants: This study was supported by JSPS KAKENHI Grant (Grant-in-Aid for Scientific Research(C)) (Number 21K08331) to Takashi Hashimoto, by “Research on Measures for Intractable Diseases”
from the Ministry of Health, Labor and Welfare Project to Takashi Hashimoto, and by grants fromTakeda Science Foundation, and AbbVie GK.
References
1. Chen KR, Shimizu S, Miyakawa S, et al. Coexistence of psoriasis and an unusual IgG-mediated subepidermalbullous dermatosis: identification of a novel 200-kDa lower lamina lucida target antigen. Br J Dermatol 1996;
134:340-346.
2. Zillikens D, Kawahara Y, Ishiko A, et al. A novel subepidermal blistering disease with autoantibodies to a 200kDa antigen of the basement membrane zone. J Invest Dermatol 1996;106:465-470.
3. Hashimoto T, Tsuruta D, Koga H, et al. Summary of results of serological tests and diagnoses for 4774 cases ofvarious autoimmune bullous diseases consulted to Kurume University. Br J Dermatol 2016;175:953-965.
4. Dainichi T, Koga H, Tsuji T, et al. From anti-p200 pemphigoid to anti-laminin gamma1 pemphigoid. J Dermatol
2010;37:231-238.
5. Goletz S, Hashimoto T, Zillikens D, et al. Anti-p200 pemphigoid. J Am Acad Dermatol 2014;71:185-191.
6. Kridin K, Ahmed AR. Anti-p200 pemphigoid: a systematic review. Front Immunol 2019;10:2466.
7. Solimani F, Pollmann R, Ishii N, et al. Diagnosis of anti-laminin γ-1 pemphigoid by immunoblot analysis. J
Eur Acad Dermatol Venereol 2019;33:735-741.
8. Dainichi T, Kurono S, Ohyama B, et al. Anti-laminin gamma-1 pemphigoid. Proc Natl Acad Sci U S A 2009;106:
2800-2805.
9. Ido H, Nakamura A, Kobayashi R, et al. The requirement of the glutamic acid residue at the third position from- 58 -IB Study of Laminin γ1 for Anti-p200 Pemphigoid
the carboxyl termini of the laminin gamma chains in integrin binding by laminins. J Biol Chem 2007;282:
11144-11154.
10. Taniguchi Y, Ido H, Sanzen N, et al. The C-terminal region of laminin beta chains modulates the integrinbinding affinities of laminins. J Biol Chem 2009;284:7820-7831.
11. Li X, Qian H, Takizawa M, et al. N-linked glycosylation on laminin γ1 inf luences recognition of anti-laminin
γ1 pemphigoid autoantibodies. J Dermatol Sci 2015;77:125-129.
12. Shimanovich I, Hirako Y, Sitaru C, et al. The autoantigen of anti-p200 pemphigoid is an acidic noncollagenous
N-linked glycoprotein of the cutaneous basement membrane. J Invest Dermatol 2003;121:1402-1408.
13. Vafia K, Groth S, Beckmann T, et al. Pathogenicity of autoantibodies in anti-p200 pemphigoid. PLoS One 2012;
7:e41769.
14. Koga H, Ishii N, Dainichi T, et al. An attempt to develop mouse model for anti-laminin γ1 pemphigoid. J DermatolSci 2013;70:108-115.
15. Florea F, Bernards C, Caproni M, et al. Ex vivo pathogenicity of anti-laminin γ1 autoantibodies. Am J Pathol
2014;184:494-506.

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Osaka City Med. J. Vol. 69, 61-65, 2023

Prolonged Olfactory Dysfunction in the COVID-19 Era;
Etiological Analysis in a Single-centered Cohort
HIROKO KAWAI1), KOUSUKE HASHIMOTO1), YUICHI TERANISHI1), YU NAKAGAMA2,3),
YASUTOSHI KIDO2,3), and KISHIKO SUNAMI1)
Departments of Otolaryngology1), Virology & Parasitology2), andResearch Center for Infectious Disease Sciences3),
Graduate School of Medicine, Osaka Metropolitan University

Abstract
Background
The prognosis of olfactory dysfunction (OD) is variable, largely depending on the etiology. The
coronavirus disease 2019 (COVID-19)-related OD tends to resolve within a few weeks; however, 10%15% of the patients suffer prolonged OD (POD) persisting beyond the first month. This study aimed
to determine the disease burden of COVID-19 in patients with undiagnosed POD and compare thecharacteristics of COVID-19-related POD with other PODs.
Methods
Patients with POD (＞4 weeks) visiting our outpatient clinic between December 2020 and January2022 were analyzed to include their clinical data, laboratory variables, endoscopy and computed
tomography findings. COVID-19 was detected using serological testing; OD severity was assessedusing the Self-Administered Odor Questionnaire, the Open Essence, and intravenous olfactometry.
Characteristics of patients with and without COVID-19 were compared.
Results
Sixteen patients (11 females; median age: 42 [15-83] years) were included, of which nine wereserologically positive for COVID-19. Compared with other etiologies, COVID-19-related POD wasmore commonly sudden onset and presented with gustatory and intranasal trigeminal dysfunction(assessed by the patientʼs inability to identify the cooling sensation elicited by menthol). Somepatients had discrepancies between the change in subjective symptoms and test results; however,most COVID-19-related POD patients showed improvement.
Conclusions
The overall burden of POD increased significantly due to the COVID-19 pandemic. Accordingly,
OD should be assessed using plural subjective tests. Further research using larger samples andtargeting different coronavirus variants is required to corroborate these results.
Key Words: ‌Olfactory dysfunction; COVID-19; Serological test
Received December 20, 2022; accepted April 11, 2023.
Correspondence to: Hiroko Kawai, MD.
Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Osaka Metropolitan
University, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
Tel: +81-6-6645-3871; Fax: +81-6-6645-0515
E-mail: c21736w@omu.ac.jp- 61 -

Kawai et al

Introduction
Prolonged olfactory dysfunction (POD) can be triggered by various etiologies, including airway
obstruction and neural disorders, although viral diseases are common cause. The recent coronavirus
disease 2019 (COVID-19) was primarily characterized by impaired olfaction, contributing to an
overall increase in the prevalence of POD; however, the exact extent is still unknown. Although
COVID-19-related olfactory dysfunction (OD) was initially considered to have a good prognosis;
resolve within a few weeks, recent long-term follow-up reports estimate a significant proportion ofpatients with COVID-19-related olfactory impairment to develop POD (1%-48%)1,2). In addition, we
supposed that there were some POD patients who were not able to receive a diagnosis, because theinspection system was insufficient just after the COVID-19 pandemic in Japan. Therefore, this study
aimed to evaluate the relative burden of COVID-19 disease in a single-center cohort of patientsdiagnosed with a POD of previously undetermined etiology. The clinical characteristics of COVID-19related POD were compared with that caused by other etiologies.

Methods
This study is a cross-sectional study, in a single cohort, which considered about the association
between the clinical characteristics and the progress of OD and COVID-19 infection. The
Strengthening the Reporting of Observation Studies in Epidemiology statement guidelines3) were
followed while reporting the findings.
Patients who visited the outpatient clinic at the Department of Otolaryngology, Osaka
Metropolitan University Hospital, Japan, between December 2020 and January 2022 for self-reported
prolonged (lasting ＞4 weeks) impairment in sense of smell were included in the study. Clinical data
regarding symptoms, laboratory variables, endoscopy and computed tomography (CT) findings, andfinal diagnosis, were extracted from medical records. This study was approved by the institutional
review board of the Osaka Metropolitan University (approval number: 2020-162). All participants
provided informed consent to participate.
To determine the etiology of their POD, participants were tested for anti-severe acute respiratory
syndrome coronavirus-2 (SARS-CoV-2) antibodies using the Elecsys Anti-SARS-CoV-2 assay (Roche,
Basel, Switzerland). Participants with positive serology against SARS-CoV-2 were diagnosed with
COVID-19-related POD. Additionally, CT was used to diagnose paranasal sinus abnormalities. Those
testing negative for SARS-CoV-2 were categorized either as non-SARS-CoV-2 post-viral POD(persistent olfactory sensation impairment after recovery from acute rhinitis) or idiopathic POD.
The pattern and severity of olfactory sense impairment were assessed in almost all patients both
qualitatively and quantitatively using the following methods: (i) Self-Administered Odor
Questionnaire (SAOQ) developed by the Japan Rhinology Society to assess an individualʼs ability toidentify 20 different odorants4). (ii) The Open Essence (OE) smell identification card to assess anindividualʼs ability to identify 12 different odorants familiar to the Japanese population5). Menthol,an OE odorant, stimulates intranasal trigeminal system. (iii) Intravenous olfactometry (IO) tomeasure the latency in recognizing garlic-like odors and perception duration after an injection ofthiamine propyl-disulfide. For the SAOQ and OE, the percentage of correct answers indicated thedegree of impairment. The participants who were available for the follow-up underwent the SAOQ or
OE tests again when more than one month has passed since the first visitation of our hospital.
Moreover, the participants underwent medical examination and interview by an otolaryngologist- 62 -

Prolonged OD in the COVID-19 Era

whenever they visited the hospital.
We compared outcomes (SAOQ, IO, and OE) between the two groups, COVID-19-related POD andnon-COVID-19-related POD. SAOQ, IO, and total point of OE were compared using Mann-Whitney U
test. The ability to recognize menthol was compared using Chi-square test. A two-sided p ＜0.05 wasconsidered statistically significant.

Results
We considered 16 patients, (11 females, 69%) with a median age of 42 years (interquartile range:
15-83 years). Nine of the 16 patients (56%) were serologically positive for COVID-19-related POD.
One participant with a ʻhigh-negativeʼ result was further tested using the Architect SARS-CoV-2 IgG
and IgG Ⅱ Quant (Abbott, Chicago, Illinois, USA) immunoassays targeting nucleocapsid and spike
proteins, respectively. In the absence of a SARS-CoV-2 vaccination, the participant had ʻpositiveʼ
results on both confirmatory immunoassays and was, thus, classified as having a positive serology.
Seven of the nine serological positive participants lacked an acute diagnosis of COVID-19. Other
causes of POD included eosinophilic sinusitis (n＝1), non-SARS-CoV-2 post-viral (n＝2), and idiopathic(n＝4). A comparison of clinical characteristics between patients of COVID-19- related POD and PODfrom other etiologies is given in Table1. In one of COVID-19-related POD patient, the SAOQ and OE
tests were not performed, and another one patient dropped out of the study.
Patients diagnosed with COVID-19-related POD were more likely to experience gustatory
dysfunction than those with an other-cause POD (n＝6/9, 67% vs n＝2/7, 29%). Although statistically
non-significant, POD severity assessed using all three methods tended to increase with COVID-19related disease severity of first assessment (median SAOQ: 8.1 vs 16.8; median OE: 4.1 vs 5.9; medianlatency of IO: 24.7 vs 14.0). Furthermore, on comparing the impairment patterns of OE odor

Table 1. Demographic and clinical variables of participants with prolonged olfactory dysfunction
COVID-19-related (n＝8) Other miscellaneous (n＝7)
Age, years (mean±SD)
Female sex, n (%)
Accompanying symptoms, n (%)
Gustation dysfunction
Dry coughNasal obstruction
None
Imaging abnormalities, n (%)
SAOQ, % (mean±SD)37.1±20.0
6 (67%)

47.7±18.6
5 (71%)

6 (67%)
1 (11%)
0 (0%)
2 (22%)
0 (0%)

2 (29%)
0 (0%)
1 (14%)
4 (57%)
1 (14%)

8.1±10.1

16.8±15.4

24.7±15.050.2±45.7

49.4±41.8

p-valuep＝0.22

IO, seconds (mean±SD)
Latency
Duration

14.0±8.2

OE, points (mean±SD)
Total

4.1±2.6

Menthol (Trigeminal dysfunction)

4 (50%)

5.9±4.4
2 (29%)

Other

3.6±2.3

5.1±4.1

Prognosis
Improvement

7 (87.5%)

3 (42.9%)

No change

1 (12.5%)

4 (57.1%)- 63 -

p＝0.36
p＝0.46p＝0.11

Kawai et al
Table 2. The clinical characteristics of COVID-19-related POD patients. Duration shows the time to first
visitation of our hospital from the onset of OD.
Sex

Age(years)

Onset

Duration(weeks)Accompany
symptoms

OE(points)

SAOQ(points)

Prognosis

Case1

Female56

After nasal
congestion

12

Gustatory
dysfunction00

Improvement

Case2

Female15

Suddenly

28

None

1

4

Improvement

Case3

Female17

Suddenly

20

Gustatory
dysfunction

5

27

Improvement

Case4

Female46

Suddenly

8

Gustatory
dysfunction

40

Improvement

Case5

Female58

Suddenly

20

Gustatory
dysfunction

8

20

Improvement

Case6

Male

16

Suddenly

16

Gustatory
dysfunction

42

Improvement

Case7

Female25

Suddenly4

Dry cough6

9Drop out

Case8

Male

35

Suddenly

8

Gustatory
dysfunction

53

Improvement

Case9

Male

66

Suddenly

56

None

None

None

No change

discrimination evaluated for different odorants, about half of the patients (n＝4) with COVID-19 were
not able to recognize the cooling sensation of menthol was higher as compared to only 29% (n＝2/7) of
those with POD from other causes. In contrast, COVID-19-related POD had a better prognosis thanother PODs (n＝7/8, 87.5% vs n＝3/4). They showed partial improvement by SAOQ, OE, and interview.
Table 2 presents details of patients with COVID-19-related POD. Eight out of nine patients with
COVID-19 POD experienced sudden onset of olfactory impairment; most of our patients were females
aged ＜40 years. The severity of olfactory impairment was variable across the patients; Case3, 4, 6,
and 8 had discrepancies between the result of OE and SAOQ, but seven of the eight patients reported
partial improvement. Case 5 and 8 received nucleic acid amplification test before the visiting our
hospital.

Discussions
In the contemporary COVID-19 era, the burden of POD of Osaka has surprisingly doubled, whichhighlights the considerable threat posed by COVID-19-related POD on the quality of life patients aswell as the Japanese public health system. Surprisingly, seven of the nine patients with COVID-19
lacked an acute diagnosis and were later diagnosed serologically, which ref lects the ground situationin Japan of a severe shortage of molecular diagnostics. Other report showed that 20% had IgG
antibodies, whereas those without a positive COVID-19 diagnosis, and olfactory or gustatory
dysfunction were the least prevalent in the population without a COVID-19 diagnosis like a symptomof fever6). The existing evidence corroborating the seroprevalence of COVID-19-related impairment inolfactory sensations used a subjective assessment of the extent of impairment and lacked quantitative
data to substantiate these findings7). Our findings confirmed the previously reported magnitude ofincreased disease burden, using robust indices of qualitative and quantitative analysis (SAOQ, OE,- 64 -

Prolonged OD in the COVID-19 Era

and IO), and medical examination by an otolaryngologist.
Like the gustatory system, the intranasal trigeminal system is also recognized as a common
neuronal target of the SARS-CoV-28). OE, widely used testing method in Japan, comprises menthol as
one of the 12 odorants used to screen any coexisting intranasal trigeminal dysfunction. Nonrecognition of the cooling sensation of menthol might indicates a trigeminal dysfunction, which wasmore commonly observed in our patients with COVID-19-related POD. It was suggested that
accompanying intranasal trigeminal dysfunction had relevance to POD 9). However, some patients
reported a subjective improvement in POD during interviews, despite an inconsistent improvement in
OE scores. This discrepancy could be attributed to the patientʼs inability to distinguish trigeminal
sensation from olfaction. Furthermore, it is difficult to evaluate olfactory dysfunction (OD) based
solely on subjective self-reporting, which was observed as the discrepancy in OE and SAOQ results ofthe same patient. Thus, multiple approaches, including diagnostic interviews, OE and SAOQ, should
be used in assessing POD clinically.
This study has some limitations. First, the sample size was small and based on single center;
therefore, we could not obtain statistically significant differences. Second, we did not differentiate
between SARS-CoV-2 variants have been reported so far with only a few differences in demographic
and symptoms. Future studies should include a large sample size and examine the differences basedon the disease variant to corroborate these results.
Acknowledgement
All authors have no COI to declare regarding the present study.
YN and YK report ownership of equity of Quantum Molecular Diagnostics, an Osaka Metropolitan
University spinout. YN and YK receive financial support outside the work from Abbott Japan LLC.
The sponsor had no control on the writing and publication of this study.
We gratefully acknowledge work of members of our laboratory and thank Enago (www.enago.jp)
for the English language review.
References
1. Paderno A, Mattavelli D, Rampinelli V, et al. Olfactory and gustatory outcomes in COVID-19: a prospective
evaluation in nonhospitalized subjects. Otolaryngol Head Neck Surg 2020;163:1144-1149.
2. Biadsee A, Dagan O, Ormianer Z, et al. Eight-month follow-up of olfactory and gustatory dysfunctions inrecovered COVID-19 patients. Am J Otolaryngol 2021;42:103065.
3. von Elm E, Altman DG, Egger M, et al. The Strengthening the Reporting of Observational Studies in
Epidemiology (STROBE) Statement: guidelines for reporting observational studies. Int J Surg 2014;12:14951499.
4. Takebayashi H, Tsuzuki K, Oka H, et al. Clinical availability of a self-administered odor questionnaire forpatients with olfactory disorders. Auris Nasus Larynx 2011;38:65-72.
5. Nishida K, Kobayashi M, Ogihara H, et al. Clinical usefulness of smell identification test card: Open Essence.
Nihon Jibiinkoka Gakkai Kaiho 2010;113:751-757. (In Japanese)
6. Bühler KM, Echeverry-Alzate V, Calleja-Conde J, et al. Seroprevalence of anti-SARS-CoV-2 IgG antibodies:
relationship with COVID-19 diagnosis, symptoms, smoking, and method of transmission. IJID Reg 2022;4:10-16.
7. Makaronidis J, Mok J, Balogun N, et al. Seroprevalence of SARS-CoV-2 antibodies in people with an acute loss
in their sense of smell and/or taste in a community-based population in London UK: an observational cohort
study. PLos Med 2020;17:e1003358.
8. Green BG. Surveying chemosensory dysfunction in COVID-19. Chem Senses 2020;45:509-511.
9. Ferreli F, Bari MD, Gaino F, et al. Trigeminal features in COVID-19 patients with smell impairment. Int
Forum Allergy Rhinol 2021;11:1253-1255.

- 65 -

Osaka City Med. J. Vol. 69, 67-74, 2023

Outcomes of COVID-19 Pneumonia Patients Complicated with
Barotrauma under Mechanical Ventilation
MASAHIRO MIYASHITA, RYO DEGUCHI, SEIYA KURIMASA, FUMIAKI WAKITA, HOSHI HIMURA,
AKIHIRO KAWAMOTO, KENICHIRO UCHIDA, TETSURO NISHIMURA,
HIROMASA YAMAMOTO, and YASUMITSU MIZOBATA
Department of Traumatology and Critical Care Medicine,
Graduate School of Medicine, Osaka Metropolitan University

Abstract
BackgroundIn the treatment of severe COVID-19 pneumonia, we often experience patients with pneumothorax
or pneumomediastinum. Complications of such barotrauma make respiratory treatments using
mechanical ventilation more complicated and difficult. The purpose of this study was to evaluate theincidence and mortality of barotrauma in patients with severe COVID-19 pneumonia and to clarify
the related factors and the effect of therapy.
Methods
From April 2020 to May 2021, 129 patients with severe COVID-19 pneumonia were treated withmechanical ventilation in the intensive care unit of the Emergency Critical Care Center of Osaka
City University Hospital. After tracheal intubation, patients were mechanically ventilated underpressure control mode that kept peak inspiratory pressure below 30 cmH2O. A muscle relaxant drug
was administered for 48 hours after tracheal intubation, and airway occlusion pressure wasmonitored to minimize patient self-inf licted lung injury. We retrospectively investigated theincidence of barotrauma and mortality of the patients.
Results
Barotrauma occurred in 11 patients (8.5%), 5 with pneumothorax alone, 4 with pneumomediastinum
alone, and 2 patients with both. The overall mortality rate of the 129 patients with severe COVID-19
pneumonia was 23%, but that of the patients complicated with barotrauma was 73%.
Conclusions
The incidence of barotrauma in severe COVID-19 pneumonia at our hospital was low comparedwith that of previous reports. Precise management of mechanical ventilation with strict preventionof patient self-inf licted lung injury might be one of the factors leading to this low incidence rate.
Key Words: ‌Barotrauma; COVID-19; Pneumonia; Pneumothorax; Pneumomediastinum
Received August 2, 2022; accepted June 13, 2023.
Correspondence to: Masahiro Miyashita, MD.
Department of Trauma and Critical Care Medicine, Graduate School of Medicine, Osaka Metropolitan
University, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
Tel: +81-6-6645-2121; Fax: +81-6-6645-3986
E-mail: miya@omu.ac.jp- 67 -

Miyashita et al

Introduction
Since the beginning of 2020, Coronavirus disease-2019 (COVID-19) pneumonia has spread rapidly
all over the world. In Japan, patients with severe COVID-19 were transferred to designated hospitals
by the government. Osaka City University Hospital, one of these designated hospitals, thus started
to treat the patients with severe COVID-19 pneumonia who required mechanical ventilation due tohypoxemia under oxygen therapy.
Patients with severe COVID-19 pneumonia mainly present with acute respiratory distresssyndrome (ARDS), and we often treat these patients with a high positive end-expiratory pressure(PEEP) ventilator setting to prevent alveolar collapse. However, increased airway pressure may
cause ventilator-related lung damage, leading to pneumothorax and pneumomediastinum. Patients
who develop such barotrauma require more careful control of airway pressure, which makes theirtreatment more difficult.
The purpose of this study was to investigate the incidence and mortality of barotrauma in patientswith severe COVID-19 pneumonia and to clarify the related factors and the effects of therapy.

Methods
We retrospectively investigated the medical records of 129 patients with severe COVID-19
pneumonia treated using mechanical ventilation at our hospital from April 2020 to May 2021. Plainchest X-rays were obtained and examined three times a week, and chest computed tomography (CT)images once a week on a routine schedule. During the first 48 hours after intratracheal intubation, amuscle relaxant was administered to suppress spontaneous breathing, and the peak inspiratory
pressure (PIP) was controlled under 30 cmH2O mainly by using the pressure control mode of themechanical ventilator. A standard tidal volume (TV) setting and airway occlusion pressure (P0.1)
monitoring were used to minimize patient self-inf licted lung injury (P-SILI). Barotrauma, such as
pneumothorax and/or pneumomediastinum, was diagnosed with the aid of plain X-ray or CT images.
Details obtained from the medical records included demographics, onset episode, onset time,
disposition, and mechanical ventilation settings (P0.1, TV, PIP). The incidence and mortality of thepatients with barotrauma were analyzed.

Results
Barotrauma occurred in 11 patients (8.5%) during the treatment for pneumonia: 5 hadpneumothorax alone, 4 had pneumomediastinum alone, and 2 patients had both (Fig. 1). The averageage of the patients who developed barotrauma was 73 years old. Most patients with pneumothorax
had an onset episode of rapid SpO 2 decline, whereas patients with pneumomediastinum werediagnosed based on slow changes in plain X-ray images, worsening respiratory status, and thepresence of subcutaneous emphysema. The average onset of barotrauma was 16 days after the onset
of COVID-19 pneumonia and 7 days after tracheal intubation. All patients with pneumothorax weremanaged with tube thoracostomy. Eight patients died and 3 patients were transferred anotherhospital to continue treatment after the acute phase of pneumonia. The overall in-hospital mortality
rate of the patients with severe COVID-19 pneumonia requiring mechanical ventilation was 23%,
whereas that of the patients with barotrauma was 73%. After the onset of barotrauma, the averagetime to death was 16 days. Bronchial asthma was observed in 2 patients and diabetes mellitus in 1
patient. None had chronic obstructive pulmonary disease, and smoking history was unavailable- 68 -COVID-19 Pneumonia with Barotrauma

Figure 1. CT images of the 68-year-old male patient with pneumothorax and pneumomediastinum. Four days aftertracheal intubation, his respiratory condition and subcutaneous emphysema worsened, and he died 6 days after the
onset of barotrauma.

(Table 1).
During the treatment period, 43 of the 129 (33%) patients had a temporary peak inspiratory
pressure exceeding 30 cmH 2O. In the patients with barotrauma, this rate was 43% before theoccurrence of barotrauma. We investigated P0.1, TV per standard body weight, and PIP during the 3days before and after the onset date. The P0.1 in all patients was less than 10 cmH2O, but it exceeded
5 cmH 2O at some points (Fig. 2). This suggested that the patients with barotrauma were notcontrolled well enough to prevent P-SILI. No obvious change of P0.1 was present before and after the
onset of the barotrauma. TV tended to decrease after onset, whereas PIP was high before and after
onset (Figs. 3 and 4). Taken together, these findings suggested that after the onset of barotrauma, it
was still difficult to control the airway pressure even if the TV was set to the minimum.
- 69 -

Miyashita et al
Table 1. Characteristics of the patients with barotrauma

Figure 2. Airway occlusion pressures during the 7 days around the onset of barotrauma. Some pressures exceed 5
cmH2O. No obvious changes are shown before and after onset.

- 70 -COVID-19 Pneumonia with Barotrauma

Figure 3. Tidal volume (TV) per standard body weight during the 7 days around the onset of barotrauma. TV tended
to decrease after onset.

Figure 4. Peak inspiratory pressure (PIP) during the 7 days around the onset of barotrauma. PIP is high at all times
around the onset date.

- 71 -

Miyashita et al

Discussion
In COVID-19 pneumonia, the virus triggers the immune system and induces the recruitment of
inf lammatory cells with subsequent production of pro-inf lammatory cytokines and chemokines. Thisphenomenon results in interstitial and alveolar edema that leads to ARDS1). Although ARDS is
managed with a high PEEP ventilator setting to prevent alveolar collapse, it is thought that the riskof alveolar damage increases due to the high peak pressure2,3). Severe and prolonged inf lammation
caused by the virus leads to the widespread destruction of alveoli and airspaces, increasing the
likelihood of rupture with slight pressure changes from mechanical ventilation 4,5). According toseveral reports, the incident of barotrauma is 2% in the patients with severe COVID-19 pneumonia
and 10 to 40% in those undergoing mechanical ventilation6-9). S. Ozdemir et al reported that among
the patients treated for COVID-19 pneumonia in the ICU, the incidence of pneumomediastinum wassignificantly higher in those treated with invasive mechanical ventilation than those treated with
non-invasive mechanical ventilation10). The rate of barotrauma at our hospital was 8.5%, which islower than that of previous reports, whereas the mortality rate of the patients with barotrauma was
73%, similar to that of other reports11-14).
We set the TV as low as possible and airway peak pressure not to exceed 30 cmH20, which is thesame as the conventional ARDS treatment, to prevent ventilator-induced lung injury 15,16) .
Furthermore, in recent years, the new concept of P-SILI has emerged, i.e., the possibility that lunginjury is induced or worsened by the patientʼs own inspiratory effort. In particular, the more severe
the lung injury is, the more frequently the trans-pulmonary pressure causes self-inf licted pulmonary
injury. It has been reported that excessive pressure on the alveolar wall due to the patientʼs
respiratory effort may induce inf lammatory cells in the lungs and promote fibrosis 17-21). At our
hospital, muscle relaxant is administered for the first 48 hours after tracheal intubation, and P0.1 ismonitored to minimize P-SILI. If P0.1 remains high after the decrease in muscle relaxation, therelaxant drug is continued for another 24 hours.
For respiratory management after barotrauma, we have no choice but to minimize TV to reduce
the airway pressure, but PIP is still high, suggesting that barotrauma makes respiratory management
even more difficult. In addition, P0.1 was relatively high in the patients with barotrauma. We cannot
determine whether this has an effect on the inability of patients with barotrauma to control P-SILI
because there is no comparative data from patients without barotrauma.
The use of steroids for patients is recommended according to the treatment of ARDS, and most of
the patients in this study also used steroids. Long-term steroid use has been implicated in alveolar
destruction, and this effect is important22-24). However, there was a difference in the amount used, andsome patients were given steroid pulses, but we thought that there was no definite trend (Table 1).
The treatment of COVID-19 pneumonia has changed significantly since the pandemic, due to viralmutation and the establishment of treatment methods. Even during the one-year period of thisstudy, there were several changes of treatments for COVID-19 pneumonia in our institution,
including the dose of steroids, antiviral drugs, and the timing of their use.
The limitations of this study are its small sample size, performance in a single facility, and lack of
a cut-off value set for P0.1. This study is just a case series study and there is no comparison data.
Therefore, we cannot definitively state how relevant these treatments were to patient outcomes. It is
undeniable that changes in treatment methods and strain mutations may have inf luenced this study.
Further study such as a large-scale comparative study is needed in the future.
- 72 -COVID-19 Pneumonia with Barotrauma

In conclusion, the incidence of barotrauma in severe COVID-19 pneumonia at our hospital was low
compared with that of previous reports. Precise management of mechanical ventilation with strict
prevention of P-SILI might be one of the factors leading to this low incidence rate.
Acknowledgements
All authors have no COI to declare regarding the present study.
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Med 2021;176:106239.
2. Brower RG, Lanken PN, MacIntyre N, et al. Higher versus lower positive end-expiratory pressures in patientswith the acute respiratory distress syndrome. N Engl J Med 2004;351:327-336.
3. Acute Respiratory Distress Syndrome Network; Brower RG, Matthay MA, et al. Ventilation with lower tidal
volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distresssyndrome. N Engl J Med 2000;342:1301-1308.
4. Ferreira JG, Rapparini C, Gomes BM, et al. Pneumothorax as a late complication of COVID-19. Rev Inst Med
Trop Sao Paulo 2020;62:e61.
5. Sun R, Liu H, Wang X. Mediastinal emphysema, giant bulla, and pneumothorax developed during the courseof COVID-19 pneumonia. Korean J Radiol 2020;21:541-544.
6. Lemmers DHL, Hilal MA, Bnà C, et al. Pneumomediastinum and subcutaneous emphysema in COVID-19:
barotrauma or lung frailty? ERJ Open Res 2020;6:00385-02020.
7. Edwards JA, Breitman I, Bienstock J, et al. Pulmonary barotrauma in mechanically ventilated coronavirus
disease 2019 patients: a case series. Ann Med Surg (Lond) 2020;61:24-29.
8. Martinelli AW, Ingle T, Newman J, et al. COVID-19 and pneumothorax: a multicentre retrospective case series.
Eur Respir J 2020;56:2002697.
9. Capaccione KM, Dʼsouza B, Leb J, et al. Pneumothorax rate in intubated patients with COVID-19. Acute Crit
Care 2021;36:81-84.
10. Özdemir S, Bilgi DÖ, Hergünsel GO, et al. Incidence and risk factors for pneumomediastinum in COVID-19
patients in the intensive care unit. Interact Cardiovasc Thorac Surg. 2022;34:236-244.
11. Elsaaran H, AlQinai S, AlTarrah D, et al. Prevalence and risk factors of barotrauma in Covid-19 patients
admitted to an intensive care unit in Kuwait; a retrospective cohort study. Ann Med Surg (Lond) 2021;63:102141.
12. Chilimuri S, Sun H, Alemam A, et al. Predictors of mortality in adults admitted with COVID-19: retrospective
cohort study from New York City. West J Emerg Med 2020;21:779-784.
13. Imam Z, Odish F, Gill I, et al. Older age and comorbidity are independent mortality predictors in a large cohortof 1305 COVID-19 patients in Michigan, United States. J Intern Med 2020;288:469-476.
14. Palaiodimos L, Kokkinidis DG, Li W, et al. Severe obesity, increasing age and male sex are independently
associated with worse in-hospital outcomes, and higher in-hospital mortality, in a cohort of patients with
COVID-19 in the Bronx, New York. Metabolism 2020;108:154262.
15. Gattinoni L, Tonetti T, Cressoni M, et al. Ventilator-related causes of lung injury: the mechanical power.
Intensive Care Med 2016;42:1567-1575.
16. Hraiech S, Dizier S, Papazian L. The use of paralytics in patients with acute respiratory distress syndrome.
Clin Chest Med 2014;35:753-763.
17. Grieco DL, Menga LS, Eleuteri D, et al. Patient self-inf licted lung injury: implications for acute hypoxemic
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18. Carteaux G, Parfait M, Combet M, et al. Patient-self inf licted lung injury: a practical review. J Clin Med 2021;
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for safe spontaneous breathing. Curr Opin Crit Care 2020;26:59-65.
20. Battaglini D, Robba C, Ball L, et al. Noninvasive respiratory support and patient self-inf licted lung injury in
COVID-19: a narrative review. Br J Anaesth 2021;127:353-364.
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Miyashita et alsteroid-induced muscle wasting. Ann Thorac Surg. 2021;112:e237-e239.
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24. Yamaya T, Baba T, Hagiwara E, et al. Pneumothorax in a COVID-19 Pneumonia patient without underlying
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- 74 -

Osaka City Med. J. Vol. 69, 75-82, 2023

A Case of Conversion Surgery for Pancreatic Head Cancer with
Liver Metastases and Renal Anemia after Combination Therapy
with Anticancer Agents and a Hypoxia-inducible Factor Prolyl
Hydroxylase Inhibitor
GO OHIRA1), RYOSUKE AMANO1), SHOGO TANAKA1), KENJIRO KIMURA1), HIROJI SHINKAWA1),
KOHEI NISHIO1), MASAHIKO KINOSHITA1), JUN TAUCHI1), DAISUKE SHIRAI2), TAKUMA OKADA2),
TAKAHITO KAWAGUCHI1), NAOKI TANI2), and TAKEAKI ISHIZAWA1)
Department of Hepatobiliary-Pancreatic Surgery1), Graduate School of Medicine,
Osaka Metropolitan University ; andDepartment of Hepatobiliary-Pancreatic Surgery2), Graduate School of Medicine,
Osaka City University

Abstract

This report describes a case of conversion surgery for pancreatic head cancer with liver metastases
and renal anemia after combination therapy with anticancer agents and a hypoxia-inducible factor
prolyl hydroxylase inhibitor.
A 70-year-old woman who visited the urology department for follow-up of postoperative bladdercancer and chronic renal failure was diagnosed with pancreatic head cancer. Computed tomography
and magnetic resonance imaging revealed no evidence of distant metastasis, and surgery was
performed. Two nodules approximately 10 mm in size were found on the liver surface and removed.
The nodules were diagnosed as metastases by rapid examination. Chemotherapy was started 4 days
after surgery. During outpatient chemotherapy, progression of renal anemia was observed, andtransfusion and erythropoietin injection were performed once, followed by Vadadustat, which made it
possible to continue chemotherapy on an outpatient basis without transfusions or erythropoietin
injections. Conversion surgery was performed 17 months after the initial diagnosis without theappearance of distant metastasis. The patient has been under observation after conversion surgery
without recurrence for approximately 7 months.
Vadadustat was safely used in a patient with pancreatic cancer and renal dysfunction, and it
effectively improved renal anemia, allowing the patient to continue outpatient chemotherapy.
Key Words: ‌Pancreatic adenocarcinoma; Conversion surgery; Renal anemia; Hypoxiainducible factor prolyl hydroxylase inhibitor; Liver metastasis

Received January 5, 2023; accepted June 13, 2023.
Correspondence to: Takeaki Ishizawa, MD, PhD, FACS.
Department of Hepatobiliary-Pancreatic Surgery, Graduate School of Medicine, Osaka Metropolitan
University, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
Tel: +81-6-6645-3840; Fax: +81-6-6646-6057
E-mail: take1438@gmail.com- 75 -

Ohira et al

Introduction
Recently, an increasing number of prospective studies including randomized controlled studies
have described the efficacy of preoperative chemotherapy for pancreatic adenocarcinoma1-3). In clinicalsituations, it is often difficult to continue the administration of effective doses of anticancer agents forpatients with renal failure, especially in the presence of refractory anemia.
A hypoxia-inducible factor prolyl hydroxylase inhibitor (HIP-PHI) is a hypoxia-inducible factor(HIF), a regulator of red blood cell production in the body, which increases the production oferythropoietin (EPO). It also induces the expression of various genes involved in iron metabolism
and suppresses hepcidin to increase iron utilization efficiency, thereby improving anemia4-6).
In this study, we report a case of pancreatic head cancer with liver metastases and chronic renal
failure associated with severe anemia that was treated by conversion pancreaticoduodenectomy after
chemotherapy (gemcitabine plus nanoparticle albumin-bound paclitaxel [GnP]) in combination with a
HIF-PHI.

Case report
In 2020, a 70-year-old woman was referred to our department from the urology department
because pancreatic head cancer was suggested by plain computed tomography (CT) (Fig. 1A) duringfollow-up for postoperative bladder cancer and chronic renal failure. Endoscopic ultrasound-guided
fine needle aspiration disclosed an adenocarcinoma lesion 20 mm in diameter in the pancreatic head(Fig. 1B). Because no vascular invasion or distant metastasis was identified by preoperative imaging
studies including magnetic resonance imaging (MRI), the patient first underwent surgery withcurative intent. At laparotomy, two nodules approximately 10 mm in size were found on hepatic
surfaces (Fig. 2), which were identified as metastatic adenocarcinoma by intraoperative rapid
diagnosis. Therefore, the surgery was completed with removal of only two nodules of the liver, andchemotherapy was considered. The Union for international cancer control (UICC) TNM 8th edition
classification at this point was T2N0M1 Stage Ⅳ7).
Because of the patientʼs poor renal function (serum blood urea nitrogen [BUN], 41 mg/dL;
creatinine [Cre]), 2.6 mg/dL; estimated glomerular filtration rate [eGFR]), 14.89 mL/min/1.7m2), GnP
was selected as the anticancer regimen. At the same time, the patient had macrocytic anemia (redblood cell, 214×104/μL; hemoglobin [Hb], 7.0 g/dL; hematocrit, 21.7%; mean corpuscular volume,
101 fL; mean corpscular hemoglobin, 32.7 pg; mean corpuscular hemoglobin consentration, 32.3% andreticulocyte count, 13.5%) that was diagnosed as renal anemia because of the lack of findings
suggesting folate/vitamin B12 deficiency (folic acid, 5.4 ng/mL; vitamin B12, 642 pg/mL) or defectiveiron utilization (Fe, 20 μg/dL, total iron binding capacity, 188 μg/dL and ferritin, 348 ng/mL).
Initially, GnP (normal regimen : gemcitabine, 1000 mg/BSA; nab-paclitaxel, 125 mg/BSA administeredon days 1, 8, and 15, every 28 days.) had been administered at 80% of the usual dose on biweekly in
combination with transfusion and EPO injection. However, renal anemia became refractory to theconventional treatment, making it difficult to continue GnP therapy on an outpatient basis after
three courses despite the rapid response in terms of tumor markers such as colorectal carcinoma
antigen 19-9 (CA19-9) and s-pancreas-1 (SPan-1)8) (Fig. 3). Therefore, we decided to administer an
HIF-PHI (vadadustat, Vafseo®, Mitsubishi Tanabe Pharma Corporation, Osaka, Japan; oral 300 mg/day) in an attempt to improve anemic conditions through the production of intrinsic erythropoietin
and improvement of iron utilization9). Vadadustat prescribed one tablet a day while checking blood- 76 -

Conversion Surgery for Pancreatic Head Cancer

Figure 1. Imaging studies.
A: Plain computed tomography before the initial surgery revealed dilatation of the main pancreatic duct (arrowheads)
and common bile duct (arrow), although the boundaries of the pancreatic tumor were unclear. No distant metastases
were suggested.
B: Endoscopic ultrasound before the initial surgery revealed a 20-mm hypoechoic nodule in the pancreatic head(arrowheads). Fine needle aspiration confirmed the presence of adenocarcinoma.

Figure 2. Liver metastases found in the initial surgery.
A yellowish-white nodule approximately 10 mm in diameter was observed on the surface of hepatic S3 beside theround ligament (arrow). Another tumor in hepatic S2 (8 mm in diameter) was confirmed only by palpation andultrasound.

samples every two weeks to keep it to the minimum necessary. Consequently, anemia was controlled
in the Hb range of 8-9 g/dL, enabling the continuation of GnP therapy after reducing the dose by 50%
and on triweekly. 14 courses of GnP (a total of 22 infusions with 6 discontinuations) were performed.
Side effects of GnP were mild hair loss and peripheral neuropathy of limbs, but chemotherapy could
be continued. Vadadustat had no noticeable side effects than mild nausea.
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Ohira et al

Figure 3. Therapeutic course of chemotherapy and the status of renal function/anemia between the two surgeries.
A hypoxia-inducible factor prolyl hydroxylase inhibitor was first administered 2 months after the start ofchemotherapy targeting Hb levels of 8-9 g/dL. In total, 12 courses of gemcitabine plus nanoparticle albumin-boundpaclitaxel (GnP) had been indicated, but the dose was reduced by 50% because of the gradual deterioration of renal
function. Tumor marker (CA19-9) levels rapidly decreased immediately after the start of GnP therapy and remainedin normal ranges until curative resection.

Figure 4. Imaging studies.
Contrast-enhanced computed tomography after chemotherapy revealed a 20-mm hypovascular tumor in the pancreatic
head (arrowheads). The dilatation of the main pancreatic duct and common bile duct identified before chemotherapy
was relieved. No metastatic lesions were demonstrated.

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Conversion Surgery for Pancreatic Head Cancer

Figure 5. Imaging studies.
A: Positron emission tomography revealed slight accumulation (SUVmax＝5) in the pancreatic head lesion (arrow). B:
No metastatic signals were visualized in the liver .

Figure 6. Histopathological images.
About 15 mm of cancer remained around the bile duct, and infiltration was observed (arrow).

Fifteen months after first operation, imaging studies including contrast-enhanced CT (Fig. 4),
MRI, and positron-emission tomography revealed no viable lesions excluding the shrunken (15 mm indiameter) primary tumor in the pancreatic head (Fig. 5). Tumor marker levels remained within normal
ranges. By contrast, the patientʼs renal dysfunction gradually progressed (serum Cre, 4.87 mg/dL;
eGFR, 7.43 mL/min/1.7m2), making it impossible to undergo further chemotherapy. Then, we offeredconversion pancreaticoduodenectomy with adjustment of acidosis with continuous hemodiafiltration.
In the second surgery, no surgical contraindications such as liver metastases and peritoneal
disseminations were found, and pancreaticoduodenectomy was completed in the usual manner. The
operative time and estimated blood loss were 378 min and 300 mL, respectively. The postoperative
course was uneventful, although hospitalization was prolonged because a shunt was created for
future hemodialysis. The pathological diagnosis of the resected specimen was Ph, mod, pT3, int,
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Ohira et al

INFb, ly0, v0, ne1, mpdx, pCH1, pDU0, pS0, pRP0, pPV0, pA0, pPL0, pOO0, pPCM0, pBCM0, pDPM0;
Evans grade Ⅲ (Fig. 6)10). Currently, the patient is alive with no evidence of recurrence 7 months
after the conversion surgery (24 months after the initial diagnosis).

Discussion
Despite recent evidence suggesting the efficacy of preoperative chemotherapy for pancreatic
adenocarcinoma, the optimal regimens for patients with renal dysfunction remain unclear. In our
department, GnP has usually been selected as a chemotherapeutic regimen in patients with renal
failure based on the fact that gemcitabine is degraded in blood to uracil metabolites, which are nottoxic to the kidneys11,12), and paclitaxel is mainly excreted into bile. In this patient, the introduction of
an HIF-PHI in place of transfusion and EPO injections played a key role in assuring the safety andfeasibility of chemotherapy on an outpatient basis . This strategy enabled 12 courses of GnP therapy,
controlling the primary cancer and possible metastatic lesions for 15 months after exploratory
laparotomy.
An HIF-PHI was first delivered to clinical practices in Japan in November 2019 as a therapeutic
drug for renal anemia during hemodialysis and then extended to patients with chronic renal failurein 2020. Vadadustat improves renal anemia by enhancing endogenous erythropoietin production
through the stabilization of HIFα enabled by its inhibitory effect on HIF prolyl hydroxylase as well as
improving iron metabolism 13,14). In addition to its pharmaceutical mechanism of action, oral
vadadustat has potential advantages over EPO injections in terms of feasibility, especially in
outpatients requiring frequent infusion treatments such as chemotherapy for malignant diseases. In
fact, some authors have suggested the efficacy of HIF-PHIs in the treatment of renal anemia duringchemotherapy for renal cancer15).
Potential concerns about the use of HIP-PHI therapy during chemotherapy include the activationof vascular endothelial growth factor, which can enhance tumor angiogenesis. In animal studies,trends of increased rates of mammary gland tumors and lung tumors were revealed, although these
events occurred following the use of an HIP-PHI at a 143-fold higher dose than the clinical dose16).
Conversely, other groups reported the inhibition of cancer progression by HIF-PHI therapy in mice,probably because of the improvements of blood f low and the oxygen distribution in cancerous tissues
because of the normalization of blood vessels17). In this patient, we planned to administer a minimaldose of an HIF-PHI to achieve Hb levels of 8-9 g/dL, aiming to avoid the potential risk of tumor
progression during chemotherapy. Other possible risks of HIF-PHI therapy in patients with cancer
undergoing curative surgery include the aggravation of diabetic retinopathy, liver dysfunction, anddevelopment of thromboembolic diseases9).
Despite recent advances in surgical techniques and chemotherapy, the survival benefit ofconversion surgery for pancreatic cancer with distant metastasis remains controversial. In previous
series of pancreatic cancer with liver metastases, favorable long-term outcomes were recorded(median overall survival, 18.2-34 months)18-22) after curative resection under conditions in which theprimary cancer and distant lesions could be controlled by chemotherapy for at least 8 months 23-25).
This patient decided to undergo curative resection because the deterioration of renal function made itdifficult to continue effective chemotherapy after 15 months of disease control. Although she hasremained alive without evidence of recurrence for 7 months after the conversion surgery, we must
follow-up this patient for years to evaluate the oncological benefit of our strategy consisting of GnP- 80 -

Conversion Surgery for Pancreatic Head Cancer

therapy in combination with HIF-PHI therapy for refractory renal anemia.
Acknowledgement
All authors have no COI to declare regarding the present study. We have obtained the informed
consent for this case report.
We thank Joe Barber Jr., PhD, from Edanz (https://www.jp.edanz.com/ac), for editing a draft of thismanuscript.
References
1. Motoi F, Kosuge T, Ueno H, et al. Randomized phase Ⅱ/Ⅲ trial of neoadjuvant chemotherapy with gemcitabine
and S-1 versus upfront surgery for resectable pancreatic cancer (Prep-02/JSAP-05). Jpn J Clin Oncol 2019;49:
190-194.
2. Birrer DL, Golcher H, Casadei R, et al. Neoadjuvant therapy for resectable pancreatic cancer: a new standardof care. pooled data from 3 randomized controlled trials. Ann Surg. 2021;274:713-720.
3. Casadei R, Di Marco M, Ricci C, et al. Neoadjuvant chemoradiotherapy and surgery versus surgery alone inresectable pancreatic cancer: a single-center prospective, randomized, controlled trial which failed to achieveaccrual targets. J Gastrointest Surg 2015;19:1802-1812.
4. Maxwell PH, Eckartdt KU. HIF prolyl hydroxylase inhibitors for the treatment of renal anaemia and beyond.
Nat Rev Nephrol 2016;12:157-168.
5. Koury MJ, Haase VH. Anaemia in kidney disease: harnessing hypoxia responses for therapy. Nat Rev Nephrol
2015;11:394-410.
6. Gupta N, Wish JB. Hypoxia-inducible factor prolyl hydroxylase inhibitors: a potential new treatment foranemia in patients with CKD. Am J Kidney Dis 2017;69:815-826.
7. Brierley J.D, Gospodarowicz M.K, Wittekind C, et al. editors. TNM Classification of Malignant Tumours. 8th ed.
Wiley-Blackwell: 2016. pp. 93.
8. Chung YS, Ho JJ, Kim YS, et al. The detection of human pancreatic cancer-associated antigen in the serum ofcancer patients. Cancer 1987;60:1636-1643.
9. Eckardt KU, Agarwal R, Aswad A, et al. Safety and efficacy of vadadustat for anemia in patients undergoing
dialysis. N Engl J Med 2021;384:1601-1612.
10. Evans DB, Rich TA, Byrd DR, et al. Preoperative chemoradiation and pancreaticoduodenectomy foradenocarcinoma of the pancreas. Arch Surg 1992;127:1335-1339.
11. Masumori N, Kunishima Y, Hirobe M, et al. Measurement of plasma concentration of gemcitabine and its
metabolite dF-dU in hemodialysis patients with advanced urothelial cancer. Jpn J Clin Oncol 2008;38:182-185.
12. Tomimaru Y, Eguchi H, Satoh T, et al. Feasibility of pre-operative chemoradiotherapy with gemcitabine totreat pancreatic cancer in patients with impaired renal function. Jpn J Clin Oncol 2015;45:343-348.
13. Stockmann C, Fandrey J. Hypoxia-induced erythropoietin production: a paradigm for oxygen-regulated gene
expression. Clin Exp Pharmacol Physiol 2006;33:968-979.
14. Yeo EJ, Cho YS, Kim MS, et al. Contribution of HIF-1alpha or HIF-2alpha to erythropoietin expression: in vivo
evidence based on chromatin immunoprecipitation. Ann Hematol 2008;87:11-17.
15. Zhou QQ, Li J, Liu B, et al. Roxadustat for treatment of anemia in a cancer patient with end-stage renal
disease: a case report. World J Clin Cases 2022;10:6587-6594.
16. Adams DF, Watkins MS, Durette L, et al. Carcinogenicity assessment of daprodustat (GSK1278863), a hypoxiainducible factor (HIF)-prolyl hydroxylase inhibitor. Toxicol Pathol 2020;48:362-378.
17. Nishide S, Matsunaga S, Shiota M, et al. Controlling the phenotype of tumor-infiltrating macrophages via the
PHD-HIF axis inhibits tumor growth in a mouse model. iScience 2019;19:940-954.
18. Sakaguchi T, Valente R, Tanaka K, et al. Surgical treatment of metastatic pancreatic ductal adenocarcinoma: areview of current literature. Pancreatology 2019;19:672-680.
19. Frigerio I, Regi P, Giardino A, et al. Downstaging in stage Ⅳ pancreatic cancer: a new population eligible forsurgery? Ann Surg Oncol 2017;24:2397-2403.
20. Wright GP, Poruk KE, Zenati MS, et al. Primary tumor resection following favorable response to systemic
chemotherapy in stage Ⅳ pancreatic adenocarcinoma with synchronous metastases: a bi-institutional analysis.
J Gastrointest Surg 2016;20:1830-1835.
21. Schneitler S, Kröpil P, Riemer J, et al. Metastasized pancreatic carcinoma with neoadjuvant FOLFIRINOX
therapy and R0 resection. World J Gastroenterol 2015;21:6384-6390.

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22. Shimura M, Mizuma M, Hayashi H, et al. A long-term survival case treated with conversion surgery following
chemotherapy after diagnostic metastasectomy for pancreatic cancer with synchronous liver metastasis. Surg
Case Rep 2017;3:132.
23. Lemke J, Barth TF, Juchems M, et al. Long-term survival following resection of brain metastases frompancreatic cancer. Anticancer Res 2011;31:4599-4603.
24. Ushida Y, Inoue Y, Oba A, et al. Optimizing indications for conversion surgery based on analysis of 454
consecutive Japanese cases with unresectable pancreatic cancer who received modified FOLFIRINOX or
gemcitabine plus nab-paclitaxel: a single-center retrospective study. Ann Surg Oncol 2022;29:5038-5050.
25. Satoi S, Yamaue H, Kato K, et al. Role of adjuvant surgery for patients with initially unresectable pancreaticcancer with a long-term favorable response to non-surgical anti-cancer treatments: results of a project study
for pancreatic surgery by the Japanese Society of Hepato-Biliary-Pancreatic Surgery. J Hepatobiliary Pancreat
Sci 2013;20:590-600.

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Osaka City Med. J. Vol. 69, 83-88, 2023

Domino Reconstruction of a Mycotic Aneurysm
in the Common Iliac Artery Using an Autologous Graftfrom the Contralateral Iliac Artery
AKIHIRO SUMIYA1), YASUYUKI BITO1), TAKANOBU AOYAMA1), MASANORI SAKAGUCHI1),YURIKO KIRIYA2), and TAKASHI MURAKAMI1)
Department of Cardiovascular Surgery1), Osaka City General Hospital; andDepartment of Cardiovascular Surgery2), Lampang Hospital

Abstract85-year-old male patient presented with a mycotic aneurysm in the right common iliac artery.
The left iliac artery was harvested beforehand and replaced with expanded polytetraf luoroethylene
graft. The mycotic aneurysm was subsequently dissected and reconstruction was performed using
autologous left iliac artery. A bypass was placed from the left external iliac artery to the right deep
femoral artery because both the right external iliac artery and right superficial femoral artery was
originally occluded. This domino reconstruction provided an autologous arterial graft for the infectedlesion, which was supposed to be more resistant to infection than any other synthetic materials.
Key Words: ‌Domino reconstruction; Autologous iliac artery graft; Mycotic aneurysm

Introduction
A mycotic aneurysm remains a serious condition that is associated with high morbidity andmortality1). An optimal treatment strategy, including the most appropriate surgical technique andmaterial, has been discussed. Here, we report the case of a patient who presented with a mycotic
aneurysm in the common iliac artery and was successfully treated by debridement and domino
reconstruction using the autologous contralateral external iliac artery and expanded
polytetraf luoroethylene (ePTFE) grafts. The patient provided consent for the writing, publication,and display of photographs in this case report.

Case Report
An 85-year-old male patient was admitted to our division for the surgical treatment of a mycotic
aneurysm in the iliac artery. A follow-up computed tomography (CT) scan for bladder cancer revealeda rapidly growing saccular aneurysm in the right common iliac artery with an irregular contour,
leading to the diagnosis of a mycotic aneurysm (Figs. 1A and 1B). A blood culture was negative forpathogens. Obstruction of the right external iliac artery (EIA) and superficial femoral artery was also
Received January 17, 2023; accepted June 13, 2023.
Correspondence to: Akihiro Sumiya, MD.
Department of Cardiovascular Surgery, Osaka City General Hospital,
2-13-22 Miyakojimahondori, Miyakojima-ku, Osaka 534-0021, Japan.
Tel: +81-6-6929-1221; Fax: +81-6-6929-3273
E-mail: aki.0809.ocum@gmail.com- 83 -

Sumiya et al

A

C

B

D

Figure 1. Preoperative computed tomographic angiogram. A saccular aneurysm with an irregular contour (arrowheads) is seen in the right common iliac artery. (A: axial, B: coronal) C, D: The right external iliac artery andsuperficial femoral artery are obstructed.

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Domino Reconstruction Using Iliac Artery

Figure 2. Schematic drawing of the surgery.

Figure 3. Computed tomographic angiogram after the surgery revealing patent autologous iliac artery (arrows) andexpanded polytetraf luoroethylene (ePTFE) grafts (green). The prosthesis graft was used for replacement of the left
external iliac artery, and the graft-to-deep femoral artery bypass was used to treat ischemia in the right extremity
ischemia.

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Sumiya et alobserved (Figs. 1C and 1D).
Prior to surgically accessing the infected lesion, a contralateral para-rectal incision was made, andan autologous graft from the left EIA was harvested via an extraperitoneal approach and replacedwith an 8-mm ePTFE graft (GORE® PROPATEN® Vascular Graft; W.L. Gore & Associates, Inc.,
Flagstaff, AZ, USA). An additional extra-anatomical bypass from the graft-to-right deep femoral
artery was constructed to treat the occluded right EIA (Fig. 2). Both grafts were placed in clean
fields, and the resulting wounds were closed before the next surgical step to prevent contamination.
A median laparotomy was subsequently performed, and intravenous heparin was administered prior
to clamping the terminal aorta and left common iliac artery. The mycotic aneurysmal sac was
opened, and the right internal iliac artery (ⅡA) was clamped using an 8 Fr balloon catheter. The
aneurysm was debrided, and the resulting tissue was sent to the pathology laboratory for culture.
Domino reconstruction from the terminal aorta to the right ⅡA was completed with the previously
harvested autologous graft.
The culture from the intraoperative debridement specimen was negative. However, histopathologyof the resected tissue was compatible with a mycotic aneurysm. Therefore, cefepime 2 g daily ̶ anempirical antibiotic ̶ was administered for 10 days. White blood cell count became normalized andC-reactive protein level reduced to 1.7 mg/dL. The patientʼs condition remained stable, and a CT scanon postoperative day 10 showed patency of both the autologous and ePTFE grafts (Fig. 3). The
patient was ambulatory and was discharged from our hospital on postoperative day 20.

Discussion
The treatment strategy for mycotic aneurysms comprises debridement of the infected tissue andreconstruction of vascular continuity. We employed an extra-anatomical crossover bypass to avoid
any foreign material in the infected region together with the use of an autologous graft to reconstruct
the right ⅡA. Patency of the extra-anatomical bypass has been reported to be slightly low. Once thegraft is occluded in the future, patency of the ⅡA will be important as a collateral to the lower
extremity. Therefore, we believed that reconstruction of the ⅡA was mandatory. Furthermore,
precluding the existence of the transected end could avoid the future formation of pseudoaneurysm.
Several materials for reconstruction have been evaluated, including in situ graft replacement
using antibiotic-coated prosthetic grafts2), cryopreserved human allografts3), autogenous femoropopliteal vein grafts4), and autologous arterial grafts5). However, the most effective material is yet to
be determined.
A cryopreserved human allograft is a good alternative to a synthetic graft for arterial
reconstruction3); however, human allografts are not easily available. The use of autologous venous
grafts (e.g., femoral, popliteal, and saphenous veins) has been reported6). Reinfection or aneurysmal
dilatation was not observed by Daenens et al6) after in situ reconstruction using the femoral veins totreat infrarenal aortic graft infections. However, serious post-surgical complications have beenreported after using grafts from the femoral or popliteal veins and include the need for revision
surgery due to major bleeding, above-the-knee amputation, graft limb occlusion, and late graft limb
stenosis4,7,8). Severe venous stasis and acute compartment syndrome were also reported after the
femoral and popliteal veins were harvested. The great saphenous vein has also been used as analternative because harvesting it is less invasive, and subsequent venous stasis is mild. To
accommodate for size discrepancies, the saphenous vein should be spliced. Reconstructions using- 86 -

Domino Reconstruction Using Iliac Artery

grafts from spiral and paneled saphenous veins are the most well-known procedures9,10). However,late aneurysmal degeneration has been reported11). These complications elucidate the limitations ofusing venous grafts for arterial reconstructions. Although autologous grafts may be tolerant toinfection, venous grafts appear to have limited integrity and durability. The use of an autologous
arterial graft would overcome these drawbacks. In our present case, an autologous graft from thecontralateral EIA was used for domino reconstruction.
Anatomical limitations of this graft included its own length and diameter. An autologous graftfrom the EIA was previously used for the reconstruction of the subclavian artery in which the sizeand length were compatible5). However, in the present case, its limited length did not allow us toanatomically bypass the occluded right EIA. Therefore, extraanatomical bypass was employed. Other
drawbacks of domino reconstruction include infection, bleeding, and potential occlusion of thesynthetic graft used to reconstruct the iliac artery where no pathology was previously present.
However, the theoretical advantages of infection resistance, durability, and the potential long-term
patency of this type of viable arterial reconstruction may outweigh these drawbacks.
In this case, ilio-deep femoral bypass was not mandatory, because ischemic symptom was minimal,such as intermittent claudication. Avoiding the use of synthetic material as possible could be a goodstrategy during infection condition. Delayed bypass for right leg ischemia could be a good option.
In conclusion, an autologous graft from the contralateral iliac artery was harvested and replacedwith an ePTFE graft. The autologous graft was used for in situ domino reconstruction to successfully
treat a mycotic aneurysm of the common iliac artery.
Acknowledgement
All authors have no COI to declare regarding the present study.
I would like to thank Shoichi Terakawa for contributing to the reconstruction and analysis of theimages.
References
1. Sörelius K, Budtz-Lilly J, Mani K, et al. Systematic review of the management of mycotic aortic aneurysms.
Eur J Vasc Endovasc Surg 2019;58:426-435.
2. Hayes PD, Nasim A, London NJ, et al. In situ replacement of infected aortic grafts with rifampicin-bonded
prostheses: the Leicester experience (1992 to 1998). J Vasc Surg 1999;30:92-98.
3. Brown KE, Heyer K, Rodriguez H, et al. Arterial reconstruction with cryopreserved human allografts in thesetting of infection: a single-center experience with midterm follow-up. J Vasc Surg 2009;49:660-666.
4. Ehsan O, Gibbons CP. A 10-year experience of using femoro-popliteal vein for re-vascularisation in graft andarterial infections. Eur J Vasc Endovasc Surg 2009;38:172-179.
5. Murakami T, Tokuda T, Nishimura S, et al. Successful treatment of Staphylococcus schleiferi infection after
aortic arch repair: in situ aortic arch replacement and domino reconstruction of the debranching graft using
autologous iliac artery. Acta Med Okayama 2020;74:251-255.
6. Daenens K, Fourneau I, Nevelsteen A. Ten-year experience in autogenous reconstruction with the femoral veinin the treatment of aortofemoral prosthetic infection. Eur J Vasc Endovasc Surg 2003;25:240-245.
7. Ali AT, Modrall JG, Hocking J, et al. Long-term results of the treatment of aortic graft infection by in situ
replacement with femoral popliteal vein grafts. J Vasc Surg 2009;50:30-39.
8. Dorweiler B, Neufang A, Chaban R, et al. Use and durability of femoral vein for autologous reconstruction with
infection of the aortoiliofemoral axis. J Vasc Surg 2014;59:675-683.
9. van Zitteren M, van der Steenhoven TJ, Burger DH, et al. Spiral vein reconstruction of the infected abdominal
aorta using the greater saphenous vein: preliminary results of the Tilburg experience. Eur J Vasc Endovasc
Surg 2011;41:637-646.
10. Mallios A, Boura B, Alomran F, et al. A new technique for reconstruction of the aortic bifurcation with- 87 -

Sumiya et alsaphenous vein panel graft. J Vasc Surg 2014;59:511-515.
11. Schwarcz TH, Nypaver TJ. Paneled saphenous vein graft repair of an iliac artery injury in a contaminatedabdomen: late complication of aneurysmal degeneration illustrating the need for continued surveillance. J
Vasc Surg 2017;66:e38-39.

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1. Priori SG, Schwartz PJ, Napolitano C, et al. Risk stratification in the long-QT syndrome. N Engl J Med
2003;348:1866-1874.
2. Schwartz PJ, Priori SG, Napolitano C. The long-QT syndrome. In: Zipes DP, Jalife J, editors. Cardiac
electrophysiology: from cell to bedside. 3rd ed. Philadelphia: W.B. Saunders; 2000. pp. 597-615.

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[Revised: June 8, 2021]

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Editorial Committee

Daisuke Tsuruta, MD (Chief Editor)
Yasuhiro Fujiwara, MD

Wakaba Fukushima, MD

Takeo Goto, MD

Yoshikazu Hiura, MD

Masayuki Hosoi, MD

Kazuo Ikeda, MD

Koki Inoue, MD

Kenichi Kohashi, MD

Yukio Miki, MD

Atsushi Shioi, MD

Toshiyuki Sumi, MD

Kishiko Sunami, MD

Junji Uchida, MD

The volumes and issues published in 1954-2018 were as follows: Vol 1 (one issue), Vols 2-5 (2issues, each), Vols 6-7 (one issue, each), Vols 8-9 (2 issues, each), Vol 10 (one issue), Vol 11 (2issues), Vol 12 (one issue), Vol 13 (2 issues), Vols 14-20 (one issue, each), Vol 21 (2 issues), Vol 22(one issue), Vols 23-65 (2 issues, each), Vol 66 (one issue), Vol 67-69 (2 issues, each)

Publisher : Osaka City Medical Association,
Graduate School of Medicine, Osaka Metropolitan University
1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan