Neural-network based high-speed volumetric dynamic optical coherence tomography.

We demonstrate deep-learning neural network (NN)-based dynamic optical coherence tomography (DOCT), which generates high-quality logarithmic-intensity-variance (LIV) DOCT images from only four OCT frames. The NN model is trained for tumor spheroid samples using a customized loss function: the weighted mean absolute error. This loss function enables highly accurate LIV image generation. The fidelity of the generated LIV images to the ground truth LIV images generated using 32 OCT frames is examined via subjective image observation and statistical analysis of image-based metrics. Fast volumetric DOCT imaging with an acquisition time of 6.55 s/volume is demonstrated using this NN-based method.

Surgical removal of multiple left ventricular thrombi with video-assisted cardioscopy: a case report.

Background: Left ventricular (LV) thrombus is a lethal complication of coronary artery disease that can lead to embolization and sudden death. There is no clear consensus on the optimal treatment for LV thrombi. There is a paucity of case series about surgical excision of LV thrombus in patients with coronary vessel disease. For that, there is insufficient evidence to support surgical excision of LV thrombus and recommend the optimal timing of this procedure. Case summary: We report a case of a 52-year-old man with a history of percutaneous catheter intervention for mid-right coronary artery lesion 3 years ago. He presented with two-vessel coronary artery disease with three LV thrombi. The thrombi were mobile and protuberant. We performed coronary artery bypass grafting in both vessels and LV thrombectomy with video-assisted cardioscopy. Discussion: Mobile or protuberant thrombus is the most important risk factor for embolization of LV thrombus. On the other hand, LV thrombus size rarely appears in studies as a risk factor for embolization, and when it does, it is a lesser risk factor. There are no case reports describing simultaneous formation of three LV thrombi after myocardial infarction, and it is not known if the risk of embolism is high in such cases. Our patient had very fragile thrombi, and thrombectomy was performed along with coronary artery bypass grafting due to the high risk of embolism.

Culture of circulating tumor cells using a microfilter device.

Circulating tumor cells (CTCs) are associated with cancer metastasis and prognosis but their scarcity in whole blood prevents their use as a diagnostic tool. The purpose of the present study was to establish a novel approach to capture and cultivate CTCs using a microfilter device. The present study was a prospective study of patients with pancreatic cancer at the University of Tsukuba Hospital (Tsukuba, Japan). From each patient, 5 ml of whole blood was collected into an EDTA collection tube. Whole blood was filtered to isolate CTCs and cells captured on the microfilter were cultured in place. A total of 15 patients were enrolled. CTCs and/or CTC clusters were detected in 2 of 6 cases on day 0. In all cases, CTCs and/or formed clusters and/or colonies were observed during long­term culture periods of up to 103 days. In samples where CTCs were not immediately evident, CTC clusters and colonies emerged after long­term culture. To confirm activity of the cultured CTCs on the filters, staining with Calcein AM was performed and epithelial cellular adhesion molecule­positive cells were observed. The system enables the capture and culture of CTCs. Cultured CTCs may be used for patient­specific drug susceptibility testing and genomic profiling of cancer.

Simultaneous Detection and HER2 Profiling of Circulating Breast Cancer Cells in Clinical Patients Using a Rare Cell Sorter.

BACKGROUND/AIM: This study describes a rare cell sorter (RCS) method to detect circulating tumor cells (CTCs) and CTC clusters in whole blood without pretreatment. PATIENTS AND METHODS: We collected samples from breast cancer patients at the University of Tsukuba Hospital. A total of 15 whole-blood specimens from patients with breast cancer were collected and analyzed via a microfluidics chip, fluorescence-conjugated antibody staining, and fluorescence microscopy. Of 15 total cases, eight were analyzed by RCS ver3 and seven were analyzed by RCS ver3.5 to reveal potential clinical differences in scanning methods. We then examined the HER2 status on 4 of the 15 patients using our RCS system. RESULTS: RCS efficiently detected all subtypes of CTCs and CTC clusters from the peripheral blood of cancer patients. The concordance rate of HER2 status between tissue and CTCs in 4 tested clinical samples was 100%. CONCLUSION: RCS is a non-invasive method that allows for simultaneous detection of CTCs, cluster presence, and surface marker (e.g., HER2) status. Frequent sampling is, thus, possible and the large amount of data obtained will be clinically useful to predict response to therapy as well as plan adjunct support therapies in cancer patients.

Results of PRospect trial to Elucidate the utility of EchocarDiography-based Cardiac ouTput in acute heart failure (PREDICT).

BACKGROUND: The predictive value of echocardiographic parameters for early worsening heart failure (WHF), worsening of symptoms of heart failure requiring intensification of therapy during an admission for acute decompensated heart failure (ADHF), has not been elucidated. METHODS: Sixteen centers in Japan prospectively enrolled 303 ADHF hospitalized patients who did not receive positive inotropic agents as an initial therapy. Physical and Doppler echocardiographic examinations were performed before the initial therapy and the association of low output findings and occurrence of early WHF were tested. RESULTS: Early WHF occurred in 63 patients with a median duration of 6 (IQR: 2-23) hours from the administration of the initial therapy. In patients with left ventricular (LV) ejection fraction <40% (N = 153), echocardiographic stroke volume index, and cardiac index showed greater c-index [95% confidence interval, 0.71 (0.61-0.79) and 0.72 (0.63-0.80), respectively] compared to single low perfusion finding (symptomatic hypotension, reduced pulse pressure, and impaired mentation) other than cool extremities. When physical findings were combined (low perfusion score), the predictive ability improved to the similar levels of echocardiographic LV output parameters [c-index: 0.69 (0.60-0.76)]. However, addition of cardiac index significantly improved the prognostic ability when added to low perfusion score [0.82 (0.74-0.87), p < 0.001]. In contrast, in patients with LV ejection fraction ≥40% (N = 150), low perfusion score, rather than LV output parameters, was predictive of early WHF [c-index: 0.73 (0.56-0.85)]. CONCLUSIONS: We demonstrated the predictive value of echocardiographic LV output indices for early WHF in ADHF patients with LV systolic dysfunction. These data support the motivation for routine use of echocardiography for initial assessment of ADHF.

Simple echocardiographic scoring in screening aortic stenosis with focused cardiac ultrasonography in the emergency department.

BACKGROUND: No established methodology exists for diagnosis of aortic stenosis (AS) using focused cardiac ultrasound (FOCUS). We evaluated the diagnostic accuracy of our developed visual AS score for screening AS in an emergency department. METHODS: Seventy-two emergency outpatients with suspected cardiovascular disease were studied. Emergency physicians assessed the visual AS score in addition to conducting the standard FOCUS, and then the aortic valve area index (AVAI) was measured by expert sonographers in the echocardiography laboratory. AVAI values >0.85 cm2/m2, 0.6-0.85 cm2/m2, and <0.6 cm2/m2 were defined as no or mild AS, moderate AS, and severe AS, respectively. RESULTS: Seventeen (24%) patients had moderate or severe AS. Visual AS scores assessed by emergency physicians and by expert sonographers showed excellent agreement (κ = 0.93), and a strong association was noted between the visual AS score assessed by emergency physicians and the AVAI assessed by expert sonographers (R = -0.71, p < 0.0001). A visual AS score ≥3 assessed by emergency physicians had a sensitivity of 82%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 95% for diagnosing moderate or severe AS. The prevalence of new-onset AS-related events during hospitalization was higher in patients with visual AS score ≥3 assessed by emergency physicians than in the remaining patients [7 (50%) vs. 2 (3%), p < 0.0001]. CONCLUSION: The visual AS score is a useful AS screening tool for emergency physicians who are not expert cardiologists.

Assessing the true severity of low-gradient aortic stenosis using resting echocardiography.

BACKGROUND: Dobutamine stress echocardiography (DSE) is required to determine whether low-gradient aortic stenosis (AS) with a small aortic valve area (AVA) is truly severe. The purpose of the present study was to evaluate the usefulness of ejection dynamics parameters at resting echocardiography for predicting the result of DSE performed in patients with low-gradient AS. METHODS: The results of resting echocardiography and DSE performed on 51 AS patients with low mean-gradient (<40 mmHg) and small indexed AVA (<0.60 cm2/m2) were retrospectively reviewed. Acceleration time (AT) and the ratio of AT to ejection time (ET) were measured on the recorded images. True-severe AS was defined as that with indexed projected AVA < 0.60 cm2/m2. RESULTS: Twenty-six (51%) patients had true-severe AS, while 22 (43%) patients had preserved left ventricular ejection fraction (≥50%). Baseline indexed AVA and AT/ET were independently associated with indexed projected AVA at DSE. AT/ET was the only independent determinant of valve compliance. Indexed AVA ≤ 0.493 cm2/m2 and AT/ET > 0.334 at baseline had sensitivities of 69% and 65% and specificities of 84% and 84%, respectively, for predicting true-severe AS. The presence of either indexed AVA ≤ 0.493 cm2/m2 or AT/ET > 0.334 had a higher sensitivity (88%), and their co-occurrence had a higher specificity (100%). CONCLUSIONS: Indexed projected AVA at DSE was predicted by AT/ET, which represented valve compliance, along with indexed AVA. The true severity of low-gradient AS can be screened using a combination of resting indexed AVA and AT/ET without performing DSE.

Mechanisms of changes in functional mitral regurgitation by preload alterations.

BACKGROUND: This study aimed to investigate the mechanisms of acute changes in functional mitral regurgitation (FMR) by preload alterations. METHODS: Twenty-two consecutive patients with left ventricular ejection fraction <40% and at least mild FMR underwent transthoracic echocardiography. Passive leg lifting and sublingual administration of nitroglycerin were performed to alter preload. Mitral regurgitant volume (MRV) was assessed using the Doppler method. RESULTS: MRV changed in parallel with preload alterations. MRV correlated better with tenting height (TH) than with mitral annular area (MAA) at baseline, whereas the difference in the correlate coefficients was not statistically significant (R=0.69 and R=0.40, respectively; p=0.19). On the other hand, changes in MRV between each sequential stage correlated better with those in MAA than with those in TH (R=0.68 and R=0.44, respectively; p=0.043). Multiple regression analysis revealed that baseline TH was the independent determinant of baseline MRV (R=0.69, p=0.0004), whereas changes in MAA with preload alteration were the independent determinant of the changes in MRV (R=0.68, p<0.0001). Changes in left atrial (LA) volume were the independent determinant of the changes in MAA (R=0.30, p=0.0063). CONCLUSIONS: Acute changes in FMR with preload alterations resulted from the transverse changes in MAA rather than the longitudinal changes in tethering-tenting of mitral geometry, and mitral annular deformation was determined by changes in LA volume. Preload reduction might help heart failure treatment through the reduction in FMR resulting from the decrease in LA and mitral annular size.

Prediction of aortic stenosis-related events in patients with systolic ejection murmur using pocket-sized echocardiography.

BACKGROUND: We have previously reported the usefulness of our newly developed visual aortic stenosis (AS) score in screening for AS using pocket-sized echocardiography. The objective of this study was to investigate whether the visual AS score and/or conventional aortic valve calcification score derived from pocket-sized echocardiography can be used to predict AS-related events. METHODS: One hundred and nine patients with systolic ejection murmur (SEM) or known AS (64 males, age 75±9 years) were enrolled and a visual AS score and an aortic valve calcification score were assessed using pocket-sized echocardiography. The primary endpoint was defined as AS-related events, including cardiac death and aortic valve replacement, during the follow-up period. RESULTS: In a multivariate Cox proportional hazards analysis, AS-related events were independently predicted by an aortic valve calcification score ≥3 (HR, 3.5; 95% CI, 1.1-11; p=0.033) and a visual AS score ≥3 (HR, 15; 95% CI, 1.8-125; p=0.013). During 18±9 months of follow-up, the event-free survival rate was 98% in patients with both a visual AS score <3 and an aortic valve calcification score <3, 90% in patients with either a visual AS score ≥3 or an aortic valve calcification score ≥3 (p<0.0001), and 62% in patients with both a visual AS score ≥3 and an aortic valve calcification score ≥3 (p<0.0001). CONCLUSIONS: The combination of visual AS score and aortic valve calcification score derived from pocket-sized echocardiography is useful for predicting AS-related events in patients with SEM.

Pre-Load-Induced Changes in Forward LV Stroke and Functional Mitral Regurgitation: Echocardiographic Detection of the Descending Limb of Starling's Curve.

OBJECTIVES: The purpose of this study was to clarify the prognostic significance of diagnosing whether the failing heart is functioning on the descending limb of the Starling curve by using echocardiography with passive leg lifting (PLL). BACKGROUND: Patients with advanced heart failure can shift to the descending limb of the Starling curve, in which pre-load does not lead to an expected increase in forward left ventricular stroke volume (LVSV). METHODS: Thirty-five consecutive patients with left ventricular (LV) ejection fraction of <40% underwent echocardiography at rest during baseline and during PLL to increase pre-load. RESULTS: Despite PLL, a paradoxical decrease in forward LVSV was observed in 15 (43%) patients. Changes in forward LVSV inversely correlated with those in functional mitral regurgitation (r = -0.56). The primary endpoint of cardiac death or hospitalization due to worsening heart failure occurred in 15 (43%) patients during follow-up (2.8 ± 2.2 years). There were a number of significant predictors of the primary endpoint in the univariate Cox analysis: baseline E/A ratio (p = 0.0002), paradoxical decrease in LVSV despite PLL (hazard ratio: 4.44; 95% confidence interval: 1.41 to 14.0; p = 0.011), baseline LV end-systolic volume (p = 0.023), and baseline LV ejection fraction (p = 0.034). In the bivariate Cox analysis, an addition of the paradoxical decrease in LVSV significantly enhanced the predictive power of all other univariate predictors. CONCLUSIONS: Heart failure patients with LV systolic dysfunction on the descending limb of the Starling curve can be recognized by the paradoxical decrease in LVSV despite PLL, and the prognostic predicting power is additive to the other traditional echocardiographic predictors. Also, our results suggest that functional mitral regurgitation is an important reason for the descending limb of the Starling curve, which is clinically recognized as the pre-load-induced decrease in forward LVSV.

Three-Dimensional Speckle Tracking Imaging for Assessing Left Atrial Function in Hypertensive Patients With Paroxysmal Atrial Fibrillation.

Hypertension (HT) is known to be the most prevalent risk factor for paroxysmal atrial fibrillation (PAF), however, its mechanisms have not been fully clarified. Our aim was to investigate the differences in left atrial (LA) function between healthy subjects, and hypertensive patients without PAF (HT-PAF(-)) and with PAF (HT-PAF(+)) using 3-dimensional (3D) speckle tracking imaging (STI). A total of 144 subjects were enrolled: 44 HT-PAF(+) (27 males; mean age 69 ± 10 years), 50 HT-PAF(-) (31 males; mean age 63 ± 11 years), and 50 controls (31 males; mean age 51 ± 14 years). All subjects were in sinus rhythm during the examination. LA volume, LA emptying fraction (LAEF), and LA wall strain were analyzed by 3D area tracking imaging. The maximal value of the global strain curve was defined as the peak global strain. The standard deviation of the time from the R-wave on the electrocardiogram to peak positive values of the segmental strain curves corrected by the R-R' interval in 6 mid LA segments (TP-SD) was calculated to assess LA dyssynchrony. LAEF and peak global strain were lower in HT-PAF(+) than in HT-PAF(-) (P < 0.01) and in the control (P < 0.01). Moreover, TP-SD was higher in HT-PAF(+) than in HT-PAF(-) (P < 0.05) and in the control (P < 0.01). Multivariate analysis revealed LA volume index, peak global strain, and TP-SD were independent determinants of HT-PAF(+). The presence of PAF is associated with diminished LA compliance and advanced mechanical dyssynchrony, as well as LA geometric deformation.

Comparison of two-dimensional and real-time three-dimensional transesophageal echocardiography in the assessment of aortic valve area.

BACKGROUND: The accuracy of two-dimensional transesophageal echocardiography (2D-TEE) for the measurement of aortic valve area (AVA) in patients with aortic stenosis (AS) depends upon the cross-section selected for imaging. Real-time three-dimensional transesophageal echocardiography (3D-TEE) may overcome this limitation of 2D-TEE. The goal of this study was to compare 3D-TEE with 2D-TEE for the measurement of AVA. METHODS AND RESULTS: Twenty-five patients with AS underwent TEE. In 2D-TEE, the aortic valve image was obtained at the orifice level in the short-axis view, and AVA was measured by planimetry of the acquired images (2D-AVA). In 3D-TEE, 3D data containing the entire aortic valve were obtained. Then, a short-axis cross-section containing the smallest orifice in mid-systole was cut from the 3D data during image postprocessing, and the AVA was measured by planimetry (3D-AVA). The 3D-AVA was significantly smaller than the 2D-AVA (0.79±0.35cm(2) vs. 0.93±0.40cm(2), p<0.0001), but there was a strong correlation between 3D-AVA and 2D-AVA (R=0.94). Although the frame rate was lower in 3D-TEE than in 2D-TEE (17±6Hz vs. 58±16Hz), the 3D-AVA determined at each frame during systole showed that the difference between 3D-AVA and 2D-AVA was not explained by the lower frame rate. The time required for image acquisition of the aortic valve was shorter with 3D-TEE than with 2D-TEE (p=0.0005). CONCLUSIONS: The geometric AVA is smaller with 3D-TEE than with 2D-TEE, and the difference is not due to the lower frame rate of 3D-TEE. The improved accuracy of 3D-TEE along with reduced image acquisition time indicates that 3D-TEE is superior to 2D-TEE for the assessment of AVA.

Antitumor effect of new HER2 peptide vaccination based on B cell epitope.

BACKGROUND: While the benefit of passive immunotherapy is commonly accepted, active immunization may have advantages for the patient's quality of life. We identified a new epitope of Mab CH401 against Her-2/neu extracellular domain (N: 167-175), and evaluated the effect of active immunization of the 20mer peptide containing the epitope (CH401 peptide). MATERIALS AND METHODS: Epitope-mapping was performed using ELISA with Her-2/neu-related multiple antigen peptides (MAP). BALB/c mice were transplanted with Her-2/neu-expressing lymphoma cell line and immunized with the peptides. For monitoring the condition, ELISA and flow cytometry was performed. RESULTS: CH401 peptide induced Her-2/neu-specific IgG antibody. Tumor growth in immunized mice was suppressed and tumor-infiltrating lymphocytes comprised more CD8(+) T-cells, which secreted larger amounts of interleukin-2 after the peptide re-stimulation. CONCLUSION: The new Her-2/neu peptide contained epitopes for CD4(+) and CD8(+) T-cells, which contributes to the suppressive effect on Her-2/neu-expressing tumor cell growth.

A novel echocardiographic index of inefficient left ventricular contraction resulting from mechanical dyssynchrony.

OBJECTIVES: The purpose of this study was to explore the possibility of using our novel echocardiographic index of inefficient left ventricular (LV) contraction in patient selection for cardiac resynchronization therapy (CRT). METHODS: Forty consecutive patients with LV ejection fraction < or = 35% were divided into 2 groups, 9 CRT candidates and 31 non-CRT candidates based on conventional criteria. A global LV time-area curve and regional LV time-area curves in 6 radial sectors were obtained using two-dimensional echocardiography in the short-axis view with speckle tracking. Fractional inefficient contraction (FIC, %) was calculated as follows: (1-global LV area change/sum of regional LV area changes) x 100. LV dyssynergy and dyssynchrony were quantified as the standard deviations of minimal values of circumferential speckle-tracking strain and their timings in the 6 sectors, respectively. RESULTS: There was no significant difference in LV dyssynchrony between CRT candidates and non-CRT candidates (79 + or - 61 ms vs. 58 + or - 26 ms, respectively). In contrast, FIC was significantly larger in CRT candidates than in non-CRT candidates (15.7 + or - 11.0% vs. 5.4 + or - 3.5%, respectively, p=0.0018), with less overlap between groups. FIC showed a positive correlation with dyssynchrony (r=0.64) and a negative correlation with dyssynergy (r=-0.42). CONCLUSIONS: Our novel echocardiographic index of inefficient LV contraction, which increases with more dyssynchrony or less dyssynergy, may prove more useful in patient selection for CRT than other indices that focus on LV temporal dyssynchrony alone.

Stenting of right coronary ostial occlusion due to thrombosed type A aortic dissection: One-year follow-up results.

A 52-year-old man experienced acute chest pain and was transferred to our hospital. An electrocardiogram showed ST-segment elevation in leads II, III, aVf, and V1 through V3. The diagnosis at the emergency room was inferior acute myocardial infarction (AMI), and emergent coronary angiography (CAG) was performed. While CAG showed subtotal occlusion of the right coronary artery (RCA) ostium, aortic dissection was suspected due to staining of the contrast agent distal to the occluded site of RCA. Intravascular ultrasound showed compression of the RCA ostium due to aortic dissection. We performed bare metal stent implantation, and contrast-enhanced computed tomography (CT) after stenting showed a thrombosed type A aortic dissection. The patient received medical treatment along with repeated CT and echocardiographic examinations, and was discharged without any events one month after admission. CAG six months after stenting and 64-multislice CT angiography one year later showed a patent RCA. Contrast-enhanced CT at six months showed complete resorption of the ascending aortic intramural hematoma, and 64-multislice CT at one year showed a descending aortic intramural hematoma. The patient is doing well one year after the onset. This is a rare case of successful medical treatment for acute type A aortic dissection complicated with AMI.

Efficient bacterial production of functional antibody fragments using a phagemid vector.

The so-called 'in vitro evolutionary method' using a phage display system has been applied for protein engineering of the antigen-binding fragment of antibodies (Fab) by conducting random mutagenesis at the antigen-binding site in combination with antigen-based biopanning. However, isolated phage clones displaying Fab cannot necessarily be used for efficient bacterial production of engineered Fab proteins, often due to deleterious defects in their proper folding abilities derived in compensation for the gain of high affinity for a particular antigen. We here report a new method of an efficient and direct bacterial expression system for the phagemid-coded Fab proteins without use of the helper phage. To overcome a low folding efficiency derived from somatic hypermutations, if any, we have established optimum conditions for bacterial cultivation and protein expression, utilizing unusually long cultivation time (>50 h) and very low temperature (25 degrees C) and thereby leading to the production and extracellular secretion of Fab proteins in a very high yield (3-15 mg/L of culture). The purified Fab folded correctly and could efficiently bind an antigen, as judged by circular dichroism and isothermal titration calorimetry, respectively.

[Primary aldosteronism with ventricular fibrillation: a case report].

A 60-year-old female had sudden onset of syncope. The emergency service noticed that she suffered cardiopulmonary arrest (ventricular fibrillation: VF). After defibrillation in the ambulance, she was transported to our emergency department. Electrocardiography monitoring showed QT prolongation. Serum potassium level was extremely low at 1.8 mEq/l. Although potassium and lidocaine were administered, it was difficult to maintain appropriate electrolyte balance and prevent VF after admission, so temporary overdrive pacing was required. She was diagnosed as having primary aldosteronism after laboratory and imaging examinations. VF was otherwise uncontrollable so a cardioverter defibrillator was implanted on the 24th hospital day. Laparoscopic adrenalglandectomy was performed about 1 month later. After the surgery, serum potassium level remained at an appropriate level without medication. No severe neurological deficits were found at discharge from our hospital.

[Efficacy of percutaneous balloon pericardiotomy and intrapericardial instillation for the management of refractory pericardial effusion: a case report].

Percutaneous balloon pericardiotomy and intrapericardial instillation seemed to be less invasive and effective treatments for refractory pericardial effusion. A 65-year-old man who suffered from refractory pericardial effusion associated with gastric cancer and had been hospitalized three times for pericardiocentesis, complained of dyspnea at rest and visited our emergency room. Echocardiography showed a large amount of pericardial effusion all around the heart and signs of cardiac tamponade. Percutaneous balloon pericardiotomy was performed and pericardial effusion turned to pleural effusion. We performed left thoracocentesis. One week later, massive pericardial effusion localized only around the right heart appeared, and pericardiocentesis was performed again. After another month, pericardial effusion around right heart appeared again and intrapericardial instillation with OK-432 (Picibanil) was tried. After the procedure, the pericardial effusion did not increase, and he has had few symptoms for 2 months as an outpatient.

Initial repertoire of anti-(4-hydroxy-3-nitrophenylacetyl) antibodies as potential donors for effective affinity maturation.

We previously found that there are two distinct antibody (Ab) maturation pathways for the immune response of C57BL/6 mice to 4-hydroxy-3-nitrophenylacetyl (NP), one involving Abs with high evolvability (group-H) and the other involving Abs with low evolvability (group-L). Commitment to whichever pathway is followed pre-determined in B cells at an early developmental stage. Candidates for the group-L or -H pathway are thus expected to pre-exist in the initial repertoire of the immune response. In the present study, we examined the initial Ab repertoire from the viewpoint of the latent potential of these Abs for effective affinity maturation. At first, we prepared anti-NP B cell hybridomas at 1 week postimmunization. Although the diversity of the obtained repertoire was maintained mainly by the third complementarity determining region of the heavy chain (CDR-H3), their changes in the near UV circular dichroism resulting from NP-binding allowed for classification into three groups according to the same rules applied in the pathway classification of the maturated Abs. This suggested that the innate structural properties of CDR-H3 were conserved throughout maturation. In other words, in exploring the structure of CDR-H3, it is possible to distinguish the latent potentials of Abs in effective affinity maturation even those making up the initial Ab repertoire. We then examined an artificially designed group-H Ab prototype and found its NP-binding ability sufficient for engagement in the initial repertoire. The question arose here as to why the majority of the actual initial repertoire consisted of the group-L ancestors regardless of their middling NP-binding affinity, which called for further discussion from the viewpoint of the dynamics possibly shaping the repertoire.

A landscape for the dynamics of an immune response.

Dynamic shaping of the antibody repertoire is essential for effective immunity. We describe here a novel approach for clarifying how the antibody repertoire is shaped over time for development of a specific immune response. We obtained over 500 immunoglobulin G1 clones harboring VH186.2 from the spleen, bone marrow, and microdissected individual germinal centers of (4-hydroxy-3-nitrophenyl) acetyl-immunized C57BL/6 mice at various time points postimmunization. Statistical analyses provided an index for defining clonal diversity and cluster analyses gave us a three-dimensional landscape with which clone distance was visualized with the expression level of antibodies. This landscape approach facilitated our understanding of the dynamics shaping the actual antibody repertoire, in which pre-existing naturally occurring population persisted and provided a significant impact upon the repertoire. To the established model for describing production of the antibody-forming cells, we were able to append an indispensable issue in considering the maturation of humoral immune response.