Strategy for affinity maturation of an antibody with high evolvability to (4-hydroxy-3-nitrophenyl) acetyl hapten

In order to quantitate the contribution of amino acid replacements to an increase in affinity during affinity maturation, we measured thermodynamic parameters of the antigen-antibody interaction for a group of anti-(4-hydroxy-3-nitrophenyl) acetyl monoclonal antibodies whose differences in amino acid sequences had arisen only from somatic hypermutation. We prepared a common ancestor and hypothetical intermediate clones that might occur on the affinity maturation pathway, by employing site-directed mutagenesis. Isothermal calorimetric titration of the antigen-antibody reaction revealed that antibody evolution proceeds in two steps. The first step is driven by a decrease in enthalpy, in which two amino acid replacements in the VL region play an essential role. Further accumulation of amino acid replacements in VH and VL regions during the second step induce a progressive increase in affinity, which is driven by an increase in entropy, which has a cooperative mutational effect.     

Intrinsic function of S100A8/A9 complex as an anti-inflammatory protein in liver injury induced by lipopolysaccharide in rats

BACKGROUND: We hypothesized that the S100A8/A9 complex is effective in the suppression of acute inflammatory changes. METHODS: To clarify such a functional role of the S100A8/A9 complex in acute inflammatory disorder, the complex purified from human leukocytes (approx. 1 mg) was intraperitoneally injected into rats 1.0 or 3.5 h after an injection of lipopolysaccharide (LPS). RESULTS: The serum concentrations of interleukin-6 (IL-6) and nitric oxide (NOx) were significantly decreased in the treated rats. Conversely, when anti-S100A8/A9 complex IgG was injected into the tail blood vessel of a rat 1.0 h after the injection of LPS, the serum concentration of IL-6 increased slightly, indicating that the antibody immunoregulatorily blocked the activity of the complex as an anti-inflammatory protein in vivo. In addition, the S100A8/A9 complex bound non-specifically with interleukin-1beta (IL-1beta), IL-6 and TNF-alpha in vitro, suggesting that the complex could bind with these cytokines in vivo. A large number of endogenous S100A8/A9 complex-positive cells that accumulated in the inflamed region in the liver 6 h after the injection of LPS were microscopically observed, while apparent inflammatory changes were not found microscopically in other organs, such as the kidney, lung and spleen. In rats treated with the S100A8/A9 complex, neither acute inflammatory changes nor S100A8/A9 complex-positive cells were also observed microscopically in the liver tissue. CONCLUSIONS: These findings suggest that the S100A8/A9 complex indirectly suppresses the overproduction of NOx from activated neutrophils and/or macrophages by neutralizing the activity of pro-inflammatory cytokines. Thus, the S100A8/A9 complex may play an important role in the suppression of acute inflammation by modulating the vital activity of pro-inflammatory cytokines in vivo.     

Vascularity of gastric carcinoma: evaluation using color Doppler ultrasonography

Purpose The purpose of this study was to investigate the vascularity of primary gastric cancer lesions using color Doppler ultrasonography.Methods We used color Doppler ultrasonography to study 78 patients with gastric cancer detected on B-mode ultrasonographic examination and 14 patients without gastric tumors but with a slightly thickened gastric wall that was also detected on B-mode ultrasound. The color Doppler signals of the gastric lesions were graded as (–), no color signals; (+), slight increase in number of color signals; and (++), an obvious increase in number of color signals. The vessel area outside the tumor area in the microscopic pathological specimens was also calculated.Results The color signals of 13 (18%) of the 71 gastric cancer patients were graded (–); those of 14 (20%) patients were graded (+); and those of 44 (62%) patients were graded (++). The color signals for 9 (65%) of 14 patients without gastric tumors were graded (–); those of 4 (28%) patients were graded (+), and those of 1 patient (7%) were graded (++). These differences were significant (P = 0.0002). The vessel count ratio in the microscopic pathologic specimens was also significantly higher in patients with an increased number of color signals than in those without an increased number of color signals (P = 0.002).Conclusion Color Doppler ultrasound showed increased vascularity in the gastric cancers in most of the subjects (82%, 58/71). Furthermore, color Doppler ultrasound also showed no increase in vascularity in most subjects (65%, 9/14) whose B-mode ultrasonograms showed thickened gastric walls but who did not have gastric cancer. Thus, color Doppler imaging may prove useful as a screening modality for gastric cancer.     

Diagnosis of gallbladder diseases by contrast-enhanced phase-inversion harmonic ultrasonography

We evaluated the usefulness of contrast-enhanced ultrasonography(US) for detecting and differentiating gallbladder lesions. Contrast-enhanced coded phase-inversion harmonic US was performed on 90 patients with gallbladder abnormalities. After administering Levovist, we observed the gallbladders in real time. Contrast-enhanced coded phase-inversion harmonic ultrasonography was compared with B-mode US and contrast-enhanced computer tomography (CT) with regard to the sensitivity and specificity in depicting the elevated gallbladder lesions. Furthermore, we assessed how the vascular patterns of the elevated gallbladder lesions depicted by contrast-enhanced US correlated with the diagnosis. Contrast-enhanced US efficiently discriminated true lesions from biliary sludge, unlike B-mode US. Consequently, contrast-enhanced US was more specific (100%) than B-mode US (81%), although their sensitivities were similar (98% and 96%, respectively). Contrast-enhanced US was also more sensitive that contrast-enhanced CT (98% versus 79%), although the two methods were equally sensitive (100% versus 95%). We classified the vascular patterns of the abnormalities depicted by contrast-enhanced US in the 90 cases into types 1 to 4, which represent branch-like, heterogeneous, homogeneous, and avascular patterns, respectively. All type 1 and 2 lesions were over 10 mm in size while most (88%) type 3 lesions were 10 mm or less in size. While the majority of carcinomas (86%) were type 1 or 2, three benign lesions also showed these patterns. Thus, the vascular pattern may simply reflect the size of the lesion and therefore its usefulness in diagnosing gallbladder lesions may be limited. Nevertheless, contrast-enhanced US is clearly superior to the other techniques in discriminating biliary sludge from other lesions.     

Duodenal ulcer penetration into the superior mesenteric artery after percutaneous transluminal angioplasty and stent placement for acute mesenteric ischemia: report of a case

A 78-year-old male presented with the chief complaints of abdominal pain and vomiting. Contrast-enhanced computed tomography and abdominal angiography showed occlusion of the superior mesenteric artery due to thrombosis, and emergency percutaneous transluminal angioplasty and stent placement were carried out. Two months later, stent thrombosis developed, and a second stent was placed. Eight months later, he complained of general fatigue and anorexia. Gastrointestinal endoscopy revealed a duodenal ulcer at the third portion close to the superior mesenteric artery. Thirteen days after conservative management, duodenal ulcer penetration into the superior mesenteric artery with subsequent air embolism developed, and the patient died of multiple organ failure.