Pitfalls and tips for lumenless lead extraction inserted deep within the ventricular septum.

Key Clinical Message: This case highlights the pitfalls and provides tips for the extraction of deeply implanted lumenless leads, and encourages careful lead selection in the current era of widespread left bundle branch area pacing. Abstract: The extraction of cardiovascular implantable electronic device leads is sometimes complicated. We describe a case with difficult but successful extraction of SelectSecure, a lumenless permanent pacemaker lead, implanted deep in the ventricular septum, highlighting its pitfalls and tips in the current era of left bundle branch area pacing.

The impact of home electrocardiograph measurement rate on the detection of atrial fibrillation recurrence after ablation: A prospective multicenter observational study.

Background: This multicenter prospective observational study examined the impact of additionally using a home electrocardiograph (ECG) to detect atrial fibrillation (AF) recurrence after ablation. Methods: Between May 2019 and December 2020, 128 patients undergoing ablation were enrolled in the study. After performing ablation, they were instructed to measure their ECGs at home using Complete (ECG paired with a blood pressure monitor; Omron Healthcare, Japan) every day and to visit the hospital every 3 months until after 12 months for 24-hour Holter ECG and 12-lead ECG as usual care (UC). Results: After ablation, 94 patients were followed up, and AF recurrence at 12 months was detected more commonly in adjudicators-interpreted Complete (31 [33 %]) than in UC (18 [9 %]) (hazard ratio 1.95, 95 % confidence interval [95 %CI] 1.35-2.81, P < 0.001). In patients with recurrent AF found via both modalities (n = 16), the time to first AF detection by Complete was 40.9 ± 73.9 days faster than that in UC (P = 0.04). Notably, when the adherence to Complete measurement was divided by 80 %, the add-on effect of Complete on the detection of recurrent AF in UC indicated the hazard ratio (HR) of 1.71 (95 %CI 0.92-3.18, P = 0.09) for the low adherence (<80 %) group, but it was significant for the high adherence (≥80 %) group, with HR of 2.19 (95 %CI 1.43-3.36, P < 0.001). Conclusions: Despite a shorter measurement time, Complete detected recurrent AF more frequently and faster compared with UC after AF ablation. A significant adherence-dependent difference of Complete was found in detecting AF recurrence.

Comparison of maximum-sized visually guided laser balloon and cryoballoon ablation.

Balloon ablation therapy has recently been used for atrial fibrillation (AF) ablation. Laser balloons possess the property in which the balloon size can be changed. Standard laser balloon ablation (Standard LBA) was followed by additional ablation using a maximally extended balloon (Extended LBA) and its lesion characteristics were compared to cryoballoon ablation (CBA), another balloon technology. From June 2020 to July 2021, patients with paroxysmal AF who underwent an initial pulmonary vein (PV) isolation were enrolled. Sixty-five patients with paroxysmal AF were included, 32 in the LBA and 33 in the CBA group. To measure the isolated surface area after the ablation procedures, left atrial voltage mapping was performed after Standard LBA, Extended LBA, and CBA. The baseline patient characteristics did not differ between LBA and CBA. Extended LBA could successfully increase the isolated area more than Standard LBA for all four PVs. Compared to CBA, the isolated area of both superior PVs was significantly greater with Extended LBA (left superior PV: 8.5 ± 2.1 vs 7.3 ± 2.4, p = 0.04, right superior PV: 11.4 ± 3.7 vs 8.7 ± 2.7, p < 0.01), and thus the non-isolated posterior wall (PW) was smaller (8.5 ± 3.4 vs 12.4 ± 3.3, p < 0.01). Nevertheless, changes in the cardiac injury markers were significantly lower with LBA than CBA. There was no significant correlation between the cardiac injury level and isolated area in both groups. In conclusion, Extended LBA exhibited a significantly greater isolation of both superior PVs and resulted in a smaller non-isolated PW, but the cardiac injury markers were significantly suppressed as compared to CBA.

Screening for untreated atrial fibrillation in the elderly population: A community-based study.

BACKGROUND: Strokes are common in people with atrial fibrillation (AF), and can have devastating consequences, especially in the elderly and if AF is untreated. However, community-based studies on screening for untreated AF have not been conducted in Japan, and there has been no evaluation of the effectiveness of early screening for AF in the elderly (≥65 years). METHODS: The Kyoto Prefectural University of Medicine (KPUM) Education Initiative has conducted an AF awareness campaign consisted of screening tests using a blood pressure (BP) monitor with electrocardiogram (ECG) (the Complete, Omron Healthcare Co., Ltd., Kyoto, Japan) and educational lectures for the elderly (≥65 years) from 2019 to 2020. A modeled effectiveness analysis was performed comparing the life-years and QALYs (quality-adjusted life-years) between direct-acting oral anticoagulation (DOAC)-treated AF and untreated AF in a Japanese setting. The basic description of the Markov model was used for the analysis. RESULTS: A total of 1648 participants were screened, and after excluding those with missing information or data (n = 41), 1607 were finally enrolled. The mean (± standard deviation) age of participants was 72.4±5.8 years, 827 (51.5%) were female, 628 (39.1%) had hypertension, and 1368 (85.1%) had CHA2DS2-VASc score ≥2. After cardiologists' evaluation of all ECG recordings of the Complete, 15 (0.93%) AF were newly detected. For each AF treated with DOAC, 0.859 QALYs gained over the lifetime for 65 years-old men, and 0.856 QALYs for 65 years-old women compared to non-treatment. CONCLUSION: A moderate number of untreated AF were identified in the community-based study. Identification of an increased number of patients with AF, if properly treated with DOAC, ultimately leads to a reduction in the number of strokes occurred over subjects' lifetime.

Depression and quality of life in older adults with atrial fibrillation: A cross-sectional community-based study.

AIM: In the older population, depression often occurs alongside physical illness. A cross-sectional community-based study examined the relationship between atrial fibrillation (AF), depression, and quality of life (QoL), and the impact of chest symptoms. METHODS: A total of 1364 older adults (≥65 years) who attended AF awareness symposia from July 2019 to December 2020 provided consent to participate in the study and valid questionnaire responses. Depression was assessed with the Geriatric Depression Scale (GDS)-15, and QoL with the 12-item Short-Form Health Survey (SF-12). RESULTS: AF patients (n = 130) were divided into symptomatic AF (n = 87) and asymptomatic AF (n = 43) groups. Depressive state and physical component summary (PCS) and mental component summary (MCS) in the SF-12 were compared with the control group (non-AF group without chest symptoms, n = 911), extracted from the same symposium participants. The depression rate (defined as GDS-15 ≥ 10) was 9.2% in symptomatic AF patients, 2.3% in asymptomatic AF patients, and 2.7% in controls. The mean PCS and MCS in each group were 43.4 ± 10.8 and 54.8 ± 8.6, 44.6 ± 10.7 and 57.3 ± 7.3, and 48.5 ± 7.9 and 56.7 ± 6.8, respectively. Multivariate regression analysis showed that symptomatic AF patients had a higher risk of depression (odds ratio: 3.46, 95% confidence interval (CI) 1.27-8.57, P = 0.01) and poor QoL (PCS: B -3.17, 95% CI -5.05 to -1.29, P = 0.001 and MCS: B -1.80, 95% CI -3.45 to -0.16, P = 0.03) compared with controls, but asymptomatic AF patients did not. CONCLUSIONS: In a cross-sectional community-based study, symptomatic AF patients were vulnerable to depression and poor QoL, but asymptomatic AF patients were not. Geriatr Gerontol Int 2022; 22: 505-510.

A Smartphone App to Improve Oral Anticoagulation Adherence in Patients With Atrial Fibrillation: Prospective Observational Study.

BACKGROUND: Poor adherence to oral anticoagulation in elderly patients with atrial fibrillation (AF) has been shown to negatively impact health care costs, morbidity, and mortality. Although various methods such as automated reminders, counseling, telephone support, and patient education have been effective in improving medication adherence, the burden on health care providers has been considerable. Recently, an attempt has been made to improve medication adherence without burdening health care providers by using smartphone apps; however, the use of the app for elderly patients with AF is still limited. OBJECTIVE: The purpose of this study was to determine whether the newly developed smartphone app for patients with AF (the Smart AF), which integrates education, automatic reminder, and patient engagement strategies with a simple user interface, can improve medication adherence in elderly patients with AF. METHODS: Patient enrollment was carried out by obtaining informed consent from patients with AF attending Kyoto Prefectural University of Medicine hospital between May 2019 and September 2020. Follow-up was planned at 1, 3, and 6 months after enrollment, and questionnaire reminders were automatically sent to patient apps at designated follow-up time points. A questionnaire-based survey of medication adherence was performed electronically using the self-reported 8-item Morisky Medication Adherence Scale (MMAS-8) as the survey tool. RESULTS: A total of 136 patients with AF were enrolled in this study. During the follow-up period, 112 (82%) patients underwent follow-up at 1 month, 107 (79%) at 3 months, and 96 (71%) at 6 months. The mean age of the enrolled patients was 64.3 years (SD 9.6), and male participants accounted for 79.4% (108/136) of the study population. The mean CHADS2 (congestive heart failure, hypertension, age, diabetes, previous stroke, or transient ischemic attack) score was 1.2, with hypertension being the most common comorbidity. At the time of enrollment, 126 (93%) and 10 (7%) patients were taking direct oral anticoagulants and warfarin, respectively. For medication adherence as measured according to the MMAS-8, MMAS scores at 1 month, 3 months, and 6 months were significantly improved compared with baseline MMAS scores (all P values less than .01). The overall improvement in medication adherence achieved by the 6-month intervention was as follows: 77.8% (14/18) of the patients in the high adherence group (score=8) at baseline remained in the same state, 45.3% (24/53) of the patients in the medium adherence group (score=6 to <8) at baseline moved to the high adherence group, and 72% (18/25) of the patients in the low adherence group (score <6) moved to either the medium or high adherence group. CONCLUSIONS: The Smart AF app improved medication adherence among elderly patients with AF. In the realm of medication management, an approach using a mobile health technology that emphasizes education, automatic reminder, and patient engagement may be helpful.

Relationships between Slc1a5 and Osteoclastogenesis.

Slc1a5 (ASCT2) encodes a small neutral amino-acid exchanger and is the most well-studied glutamine transporter in cancer cells. To investigate the role of Slc1a5 in osteoclastogenesis, we developed Slc1a5-deficient mice by using a conventional gene-targeting approach. The Slc1a5-/- mice showed no obvious abnormalities in growth. Glutamine uptake was assessed in Slc1a5+/+ and Slc1a5-/- bone marrow cells stimulated with RANKL. The rate of glutamine uptake in Slc1a5-/- bone marrow cells was reduced to 70% of that of cells from Slc1a5+/+ bone marrow. To confirm the involvement of Slc1a5 in osteoclast formation, bone marrow cells derived from Slc1a5+/+ or Slc1a5-/- mice were stimulated with RANKL and macrophage colony-stimulating factor and stained with tartrate-resistant acid phosphatase. The bone resorption activity and actin ring formation of stimulated cells were measured. The formation of multinucleated osteoclasts in bone marrow cells isolated from Slc1a5-/- mice was severely impaired compared with those from Slc1a5+/+ mice. RANKL-induced expression of ERK, NFκB, p70S6K, and NFATc1 was suppressed in Slc1a5-/- osteoclasts. These results show that Slc1a5 plays an important role in osteoclast formation.

Immortalization of human hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing.

Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

Importance of Preoperative Computed Tomography Assessment of the Membranous Septal Anatomy in Patients Undergoing Transcatheter Aortic Valve Replacement With a Balloon-Expandable Valve.

BACKGROUND: Cardiac conduction disturbance (CD) is the most frequent complication following transcatheter aortic valve replacement (TAVR). This study examined whether the anatomy of the membranous septum (MS) could provide useful information about the risk of CD following TAVR with a balloon-expandable valve (BEV).Methods and Results:Among 132 consecutive patients, 106 (mean age, 85.6±5.1 years; 75 females) were included in the study. Using preoperative CT and angiography, MS length and implantation depth (ID) were assessed. The MS length minus the prosthesis ID was calculated (∆MSID). Correlation between CD, defined as new-onset left-bundle branch block (LBBB) or the need for permanent pacemaker (PPM) within 1 week after the procedure, and MS length were evaluated. A total of 19 patients (18%) developed CD following TAVR. MS length was significantly shorter in these patients than in those without CD (5.3±1.3 vs. 6.6±1.4; P<0.001), and was the important predictor of CD (odds ratio [OR]: 0.43, 95% confidence interval [CI]: 0.27-0.69, P<0.001). When considering the pre- and postprocedural parameters, the ∆MSID was smaller in patients with CD (-1.7±1.5 vs. 0.8±1.9, P<0.001), and emerged as the important predictor of CD (OR: 0.47, 95% CI: 0.33-0.69, P<0.001). CONCLUSIONS: Short MS is associated with an increased risk of CD after TAVR with BEV.

UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase is indispensable for oogenesis, oocyte-to-embryo transition, and larval development of the nematode Caenorhabditis elegans.

N-linked glycosylation of proteins is the most common post-translational modification of proteins. The enzyme UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) catalyses the first step of N-glycosylation, and DPAGT1 knockout is embryonic lethal in mice. In this study, we identified the sole orthologue (algn-7) of the human DPAGT1 in the nematode C. elegans. The gene activity was disrupted by RNAi and deletion mutagenesis, which resulted in larval lethality, defects in oogenesis and oocyte-to-embryo transition. Endomitotic oocytes, abnormal fusion of pronuclei, abnormal AB cell rotation, disruption of permeation barriers of eggs, and abnormal expression of chitin and chitin synthase in oocytes and eggs were the typical phenotypes observed. The results indicate that N-glycosylation is indispensable for these processes. We further screened an N-glycosylated protein database of C. elegans, and identified 456 germline-expressed genes coding N-glycosylated proteins. By examining RNAi phenotypes, we identified five germline-expressed genes showing similar phenotypes to the algn-7 (RNAi) animals. They were ribo-1, stt-3, ptc-1, ptc-2, and vha-19. We identified known congenital disorders of glycosylation (CDG) genes (ribo-1 and stt-3) and a recently found CDG gene (vha-19). The results show that phenotype analyses using the nematode could be a powerful tool to detect new CDG candidate genes and their associated gene networks.

Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells.

The potential applications of human embryonic stem cells (hESCs) in regenerative medicine and developmental research have made stem cell biology one of the most fascinating and rapidly expanding fields of biomedicine. The first clinical trial of hESCs in humans has begun, and the field of stem cell therapy has just entered a new era. Here, we report seven hESC lines (SEES-1, -2, -3, -4, -5, -6, and -7). Four of them were derived and maintained on irradiated human mesenchymal stem cells (hMSCs) grown in xenogeneic-free defined media and substrate. Xenogeneic-free hMSCs isolated from the subcutaneous tissue of extra fingers from individuals with polydactyly showed appropriate potentials as feeder layers in the pluripotency and growth of hESCs. In this report, we describe a comprehensive characterization of these newly derived SEES cell lines. In addition, we developed a scalable culture system for hESCs having high biological safety by using gamma-irradiated serum replacement and pharmaceutical-grade recombinant basic fibroblast growth factor (bFGF, also known as trafermin). This is first report describing the maintenance of hESC pluripotency using pharmaceutical-grade human recombinant bFGF (trafermin) and gamma-irradiated serum replacement. Our defined medium system provides a path to scalability in Good Manufacturing Practice (GMP) settings for the generation of clinically relevant cell types from pluripotent cells for therapeutic applications.

Infection of the upper respiratory tract of hamsters by the bovine parainfluenza virus type 3 BN-1 strain expressing enhanced green fluorescent protein.

Bovine parainfluenza virus type 3 (BPIV3) is an important pathogen associated with bovine respiratory disease complex (BRDC). We have generated a recombinant BPIV3 expressing enhanced green fluorescent protein (rBPIV3-EGFP) based on the BN-1 strain isolated in Japan. After intranasal infection of hamsters with rBPIV3-EGFP, EGFP fluorescence was detected in the upper respiratory tract including the nasal turbinates, pharynx, larynx, and trachea. In the nasal turbinates, rBPIV3-EGFP attained high titers (>10(6) TCID50/g of tissue) 2-4 days after infection. Ciliated epithelial cells in the nasal turbinates and trachea were infected with rBPIV3-EGFP. Histopathological analysis indicated that mucosal epithelial cells in bronchi were shed by 6 days after infection, leaving non-ciliated cells, which may have increased susceptibility to bacterial infection leading to the development of BRDC. These data indicate that rBPIV3-EGFP infection of hamsters is a useful small animal model for studying the development of BPIV3-associated BRDC.

Complete genome sequence of the first isolate of genotype C bovine parainfluenza virus type 3 in Japan.

Bovine parainfluenza virus type 3 (BPIV3) isolates are classified into three genotypes (BPIV3a to -c). Here, we report the complete genome sequence of the BPIV3c isolate for the first time in Japan. Our results indicate that new primer sets will be required to detect all genotypes of BPIV3 strains.

Glycoproteomics approach for identifying Glycobiomarker candidate molecules for tissue type classification of non-small cell lung carcinoma.

Histopathological classification of lung cancer has important implications in the application of clinical practice guidelines and the prediction of patient prognosis. Thus, we focused on discovering glycobiomarker candidates to classify the types of lung cancer tissue. First, we performed lectin microarray analysis of lung cancer tissue specimens and cell lines and identified Aleuria aurantia lectin (AAL), Hippeastrum hybrid lectin (HHL), and Concanavalia ensiformis agglutinin (ConA) as lectin probes specific to non-small cell lung carcinoma (NSCLC). LC-MS-based analysis was performed for the comprehensive identification of glycoproteins and N-linked glycosylation sites using lectin affinity capture of NSCLC-specific glycoforms of glycoproteins. This analysis identified 1092 AAL-bound glycoproteins (316 gene symbols) and 948 HHL/ConA-bound glycoproteins (279 gene symbols). The lectin microarray-assisted verification using 15 lung cancer cell lines revealed the NSCLC-specific expression of fibronectin. The glycosylation profiling of fibronectin indicated that the peanut agglutinin (PNA) signal appeared to differentiate two NSCLC types, adenocarcinoma and large cell carcinoma, whereas the protein expression level was similar between these types. Our glycoproteomics approach together with the concurrent use of an antibody and lectin is applicable to the quantitative and qualitative monitoring of variations in glycosylation of fibronectin specific to certain types of lung cancer tissue.

Determination of glycan motifs using serial lectin affinity chromatography.

Serial lectin affinity chromatography is a convenient technique for characterizing glycan motifs (terminal glycan structures) of glycoproteins or released glycans. When these glycoconjugates are applied serially or in parallel to lectin-immobilized columns, information regarding the glycan motifs can be obtained. We demonstrate lectin affinity chromatographic methods for determining O-linked glycan structures of MUC1 purified from a breast cancer cell line, YMB-S, N-linked glycan structures of serum prostate-specific antigen from prostate cancer, and serum alkaline phosphatases from choriocarcinoma. These lectin-fractionated samples are analyzed quantitatively by measuring radioactivity, antigen contents are analyzed using enzyme-linked immunosorbent assay, and enzymatic activities are assessed.

Influenza A virus hemagglutinin and neuraminidase mutually accelerate their apical targeting through clustering of lipid rafts.

UNLABELLED: In polarized epithelial cells, influenza A virus hemagglutinin (HA) and neuraminidase (NA) are intrinsically associated with lipid rafts and target the apical plasma membrane for viral assembly and budding. Previous studies have indicated that the transmembrane domain (TMD) and cytoplasmic tail (CT) of HA and NA are required for association with lipid rafts, but the raft dependencies of their apical targeting are controversial. Here, we show that coexpression of HA with NA accelerated their apical targeting through accumulation in lipid rafts. HA was targeted to the apical plasma membrane even when expressed alone, but the kinetics was much slower than that of HA in infected cells. Coexpression experiments revealed that apical targeting of HA and NA was accelerated by their coexpression. The apical targeting of HA was also accelerated by coexpression with M1 but not M2. The mutations in the outer leaflet of the TMD and the deletion of the CT in HA and NA that reduced their association with lipid rafts abolished the acceleration of their apical transport, indicating that the lipid raft association is essential for efficient apical trafficking of HA and NA. An in situ proximity ligation assay (PLA) revealed that HA and NA were accumulated and clustered in the cytoplasmic compartments only when both were associated with lipid rafts. Analysis with mutant viruses containing nonraft HA/NA confirmed these findings. We further analyzed lipid raft markers by in situ PLA and suggest a possible mechanism of the accelerated apical transport of HA and NA via clustering of lipid rafts. IMPORTANCE: Lipid rafts serve as sites for viral entry, particle assembly, and budding, leading to efficient viral replication. The influenza A virus utilizes lipid rafts for apical plasma membrane targeting and particle budding. The hemagglutinin (HA) and neuraminidase (NA) of influenza virus, key players for particle assembly, contain determinants for apical sorting and lipid raft association. However, it remains to be elucidated how lipid rafts contribute to the apical trafficking and budding. We investigated the relation of lipid raft association of HA and NA to the efficiency of apical trafficking. We show that coexpression of HA and NA induces their accumulation in lipid rafts and accelerates their apical targeting, and we suggest that the accelerated apical transport likely occurs by clustering of lipid rafts at the TGN. This finding provides the first evidence that two different raft-associated viral proteins induce lipid raft clustering, thereby accelerating apical trafficking of the viral proteins.

Glycoproteomic discovery of serological biomarker candidates for HCV/HBV infection-associated liver fibrosis and hepatocellular carcinoma.

We previously proposed a high-throughput strategy to discover serological biomarker candidates of cancer. This strategy focuses on a series of candidate glycoproteins that are specifically expressed in the original tissues (cells) of the target cancer and that carry glycan structures associated with carcinogenesis [Narimatsu, H., et al. FEBS J.2010, 277(1), 95-105]. Here, we examined the effectiveness of our strategy in identifying biomarkers to assess progression of liver fibrosis and for the early detection of hepatocellular carcinoma (HCC). On the basis of the results of lectin array analyses in culture media of hepatoma cell lines, we captured glycopeptides carrying AAL-ligands (fucosylated glycans) or DSA-ligands (branched glycans) from digests of culture media proteins and sera from HCC patients with a background of liver cirrhosis (LC). Glycoproteins were identified by the IGOT-LC-MS method. In all, 21 candidates were selected from 744 AAL-bound glycoproteins for further verification according to (i) their abundance in serum, (ii) their specific expression in liver, and (iii) the availability of antibodies to the glycoproteins. All selected candidates showed enhancement of AAL-reactivity in sera of HCC patients compared with that of healthy volunteers (HV). These results indicate that our glycoproteomic strategy is effective for identifying multiple glyco-biomarker candidates in a high-throughput manner.

Complete Genome Sequences of Bovine Parainfluenza Virus Type 3 Strain BN-1 and Vaccine Strain BN-CE.

Bovine parainfluenza virus type 3 (BPIV3) is associated with upper respiratory disease in cattle in many countries. Here, we report the complete genome sequences of the BPIV3 BN-1 strain, isolated from cattle in Japan, and the BN-CE vaccine strain, derived from the BN-1 strain by passages in chicken embryo fibroblasts.

Epitope mapping of neutralizing monoclonal antibody in avian influenza A H5N1 virus hemagglutinin.

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.