The First Known Case of Liver Abscess Caused by Aggregatibacter aphrophilus in Japan.

A 48-year-old man presented with a sustained fever. Abdominal computed tomography revealed multilocular liver abscesses. He underwent percutaneous needle aspiration, yielding straw-colored pus. Gram staining revealed Gram-negative coccobacilli. The organism grew only on chocolate II agar in a 7% carbon dioxide atmosphere. Identification of Aggregatibacter aphrophilus was confirmed using mass spectrometry and 16S rRNA gene sequencing. He was successfully treated with antibiotics. Liver abscess caused by A. aphrophilus is extremely rare. We herein report the first such case in Japan. Even fastidious organisms, such as A. aphrophilus, should be correctly identified using mass spectrometry or 16S rRNA gene sequencing for adequate treatment.

Congenital dysfibrinogenemia coincidentally diagnosed at the onset of chronic myelogenous leukemia.

A 34-year-old man was referred to our hospital for leukocytosis and fundal hemorrhage. Peripheral blood and coagulation tests showed increases in cells at all stages of the neutrophilic series and a low level of fibrinogen (Fbg). Chronic myelogenous leukemia (CML) was diagnosed, and nilotinib was administered. During the clinical course of CML treatment, plasma Fbg levels continued to be low, but the patient showed neither hemorrhagic nor thrombotic complications. Fbg analysis showed normal antigen levels and low activity levels, which indicated dysfibrinogenemia. Genetic analysis revealed a heterozygous gene mutation (γ308AAT→AAG), a mutation which was also found in the patient's mother. Asymptomatic patients with dysfibrinogenemia have a low risk of hemorrhage in daily life and do not require treatment. However, in those undergoing major surgery or in serious accidents, replacement therapy may be required. When the cause of low Fbg levels is unknown, dysfibrinogenemia or fibrinogen deficiency should be considered. Even asymptomatic patients may benefit from more detailed immunologic and genetic analyses.

Nonsense-mediated mRNA decay was demonstrated in two hypofibrinogenemias caused by heterozygous nonsense mutations of FGG, Shizuoka III and Kanazawa II.

We report two novel hypofibrinogenemias, Shizuoka III and Kanazawa II, which are caused by heterozygous mutations in FGG. Shizuoka III showed c.147delT and 147_149insACA in FGG exon 3 and a subsequent frameshift mutation, resulting in mature protein γ23X (native protein: γ49X), and Kanazawa II showed c.1205G>A in FGG exon 9, resulting in γ376X (native protein: γ402X). To determine whether the truncated γ-chains, γ23X and γ376X, were synthesized and participated in the assembly of fibrinogen, mutant-type cDNA vectors were transfected into Chinese hamster ovary (CHO) cells. Significant levels of mutant fibrinogen were not detected by ELISA in the culture media and cell lysates. Immunoblot analysis of cell lysates revealed that the mutant γ-chain of γ376X was observed but intact fibrinogen was not. On the other hand, mutant γ-chain was not observed in γ23X-expressing cells. To demonstrate the involvement of the mechanisms of nonsense-mediated mRNA decay (NMD), we cloned wild- and mutant-type mini-genes containing γ23 or γ376 codon and transfected these into CHO cell lines in the absence or presence of cycloheximide as an NMD inhibitor. mRNA levels were determined using real-time quantitative RT-PCR in CHO cells. In the absence of cycloheximide, levels of mRNAs transcribed from the mutant gene were lower than from the wild-type gene whereas, in the presence of cycloheximide, levels of mRNAs transcribed from the mutant gene increased dose-dependently. Finally, these results demonstrated that mRNAs containing γ23X or γ376X are degraded by the NMD system and translation of the truncated γ-chain polypeptide decrease in patients' hepatocytes, resulting in hypofibrinogenemias.

Fibrinopeptide A release is necessary for effective B:b interactions in polymerisation of variant fibrinogens with impaired A:a interactions.

Fibrin polymerisation is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b'. We demonstrated markedly delayed thrombin-catalysed fibrin polymerisation, through B:b interactions alone, of recombinant γD364H -fibrinogen with impaired hole 'a'. To determine whether recombinant variant fibrinogens with no release of fibrinopeptide A (FpA) polymerise similarly to γD364H -fibrinogen, we examined two variant fibrinogens with substitutions altering knob 'A', Aα17A- and Aα17C-fibrinogen. We examined thrombin- or batroxobin-catalysed fibrinopeptide release by HPLC, fibrin clot formation by turbidity and fibrin clot structure by scanning electron microscopy (SEM) and compared the results of the variants with those for γ D364H-fibrinogen. Thrombin-catalysed FpA release of Aα17A-fibrinogen was substantially delayed and none observed for Aα17C-fibrinogen; fibrinopeptide B (FpB) release was delayed for all variants. All variant fibrinogens showed substantially impaired thrombin-catalysed polymerisation; for Aα17A-fibrinogen it was delayed less, and for Aα17C more than for γD364H -fibrinogen. No variants polymerised with batroxobin, which exposed only knob 'A'. The inhibition of variant fibrinogens' polymerisation was dose-dependent on the concentration of either GPRP or GHRP, and both peptides that block holes 'b'. SEM showed that the variant clots from Aα17A- and γD364H-fibrinogen had uniform, ordered fibres, thicker than normal, whereas Aα17C -fibrinogen formed less organised clots with shorter, thinner, and tapered ends. These results demonstrate that FpA release per se is necessary for effective B:b interactions during polymerisation of variant fibrinogens with impaired A:a interactions.

[Functional analysis for dysfibrinogenemias, Toyama and Adachi, which have a mutation of Aalpha16Arg-->His (CGT-->CAT) with aberrant fibrinopeptide A release].

We found and identified four heterozygous dysfibrinogenemias with AalphaR16H(CGT-->CAT) mutation in two families by coagulation tests and direct sequence analysis for PCR-amplified DNA fragments. Two dysfibrinogens were designated as fibrinogen Toyama and Adachi, according to the place of residence of proposituses, respectively. Patients' fibrinogen purified from plasma using immunoaffinity-chromatography was subjected to thrombin- or batroxobin-catalyzed fibrin polymerization, fibrinopeptide A (FPA) release, and clottability test. AalphaR16H-fibrinogen showed impaired thrombin or batroxobin-catalyzed fibrin polymerization in comparison with normal control fibrinogen. It is interesting that the period of protofibril formation of Toyama propositus was longest in those of four affected people. The clottability of AalphaR16H-fibrinogen was 66-70% with thrombin and higher than with batroxobin, 35-50%. In the same condition with fibrin polymerization, thrombin and batroxobin did not cleave the Aalpha16H-17G peptide-bonding, resulting in no release of variant FPA. From these results, we speculated that elongation of the two-stranded protofibril formation would be terminated by participation of the heterodimer fibrinogen molecules composed with a normal and an aberrant Aalpha-chain, and it would result in a decrease in fibrin polymerization. We speculated that the difference in the extent of impairment of fibrin polymerization among the patients might be caused by the different amount of heterodimers. Moreover, we also speculated that batroxobin-induced clottability was lower than thrombin-induced clottability, because batroxobin cannot induce the so-called "B-knob-b-hole" interaction, which enhances fibrin formation.