Histopathological and immunohistochemical findings of 20 autopsy cases with 2009 H1N1 virus infection.

Twenty autopsy cases with 2009 pandemic influenza A (2009 H1N1) virus infection, performed between August 2009 and February 2010, were histopathologically analyzed. Hematoxylin-eosin staining, immunohistochemistry for type A influenza nucleoprotein antigen, and real-time reverse transcription-PCR assay for viral RNA were performed on formalin-fixed and paraffin-embedded specimens. In addition, the D222G amino acid substitution in influenza virus hemagglutinin, which binds to specific cell receptors, was analyzed in formalin-fixed and paraffin-embedded trachea and lung sections by direct sequencing of PCR-amplified products. There were several histopathological patterns in the lung according to the most remarkable findings in each case: acute diffuse alveolar damage (DAD) with a hyaline membrane (four cases), organized DAD (one case), acute massive intra-alveolar edema with variable degrees of hemorrhage (three cases), neutrophilic bronchopneumonia (five cases) and tracheobronchitis with limited histopathological changes in alveoli (four cases). In two cases, the main findings were due to preexisting disease. Influenza virus antigen was only detected in the respiratory tract in 10 cases by immunohistochemistry. The antigen was detected in type II pneumocytes (three cases) in the epithelial cells of the trachea, bronchi and glands (six cases), and in the epithelial cells in both of the above (one case). The four cases with acute DAD presented with antigen-positive type II pneumocytes. In one case, the D222G substitution was detected in the lung as a major sequence, although 222D was prominent in the trachea, suggesting that selection of the viral clones occurred in the respiratory tract. In five cases, the pathogenesis of 2009 H1N1 was confirmed to be viral infection in pneumocytes, which caused severe alveolar damage and fatal viral pneumonia. Further studies on both host and viral factors in autopsy or biopsy materials will be essential to elucidate the other pathogenic factors involved in influenza virus infection.

Normal delivery following resection of an androgen-secreting adrenal carcinoma.

A 31-year-old female presented to a gynecological clinic complaining of amenorrhea and virilism over a 2-month period. Blood tests revealed high serum total testosterone and free testosterone levels. A left adrenal tumor was identified following computed tomography and she was referred to our clinic where a laparoscopic left adrenalectomy was performed. The tumor weighed 98 g and the pathological diagnosis according to Weiss' criteria was adrenocortical carcinoma. The post-operative course was uneventful; her serum free testosterone level normalized and regular menstruation was observed 1 month post-operatively. The patient became pregnant 1 year later, resulting in the normal delivery of a girl.

[Renal solitary fibrous tumor with hemangiopericytoma-like pattern--a case report].

A 67-year-old man was referred to our clinic with a complaint of macroscopic hematuria. He had been treated for schizophrenia in a psychology ward. Computed tomography (CT) scan revealed a left renal tumor 7 cm in size, well enhanced in early phase. Doppler ultrasonography indicated hypervascular left renal tumor. Laparoscopic radical left nephrectomy was performed under the diagnosis ofrenal cancer. Histopathlogical results showed a renal solitary fibrous tumor (SFT) with a hemangiopericytoma-like pattern. Renal SFT is an uncommon tumor. The present case is the 11th reported case of SFT with hemangiopericytoma-like pattern in the Japanese literature.

[Histopathological examination and analysis of mortality in DBA/2 mouse vasculitis induced with CAWS, a water-soluble extracellular polysaccharide fraction obtained from Candida albicans].

CAWS, a water-soluble extracellular polysaccharide fraction obtained from the culture supernatant of Candida albicans, is one of the fungal pathogen-associated molecular patterns (PAMPs). It has been reported to show potent activity inducing arteritis and coronaritis in mice. Especially, CAWS-induced arteritis has a 100% incidence and severe mortality in the DBA/2 mouse strain. This artificial vasculitis was reported to provide a good murine model of Kawasaki disease and other inflammatory vascular disease. However, severe mortality was observed only in DBA/2 mice, which is a CAWS-sensitive strain. In this study, to clarify the mechanisms of CAWS-induced arteritis and mortality, we investigated microscopic histopathological changes in cardiovascular tissues in DBA/2 mice. Severe inflammatory infiltration was observed from the external elastic lamina in the aorta and proximal coronary arteries within 1 week after CAWS administration. Severe stenosis of the aorta and coronary arteries was observed more than 3 weeks after CAWS administration. Fibrinoid necrosis was observed in these vessel walls. All CAWS-treated mice died between the fifth and twelfth week after administration. Severe inflammatory change with aortic valve transformation suggested that CAWS-treated mice died of valvular endocarditis or cardiac dysfunction. Based on the simple induction method and complete incidence, these data suggest that CAWS-induced arteritis is a good model of not only Kawasaki disease but also other cardiovascular diseases such as valvular endocarditis.

Intravascular large B cell lymphoma diagnosed by senile angioma biopsy.

An 82-year-old man without notable medical history was admitted to our hospital following subacute deterioration of apettite, disorientation and strange behavior. There was spasticity of the right extremities without weakness. LDH and serum soluble IL2 receptor antibody levels were elevated, and as well as the protein level and IgG levels in the cerebrospinal fluid. CT scanning of the brain revealed a lesion at the left corona radiata. The patient's level of consciousness was worsening, and follow-up study of the brain showed a new lesion in the left occipital lobe. Intravascular lymphomatosis was therefore suspected. We performed a skin biopsy from two typical senile angiomas. In one of these biopsy specimens, a capillary hemangioma was present in the mid-reticular dermis and it was filled with abnormal B cells. The diagnosis of intravascular B cell lymphoma (IVL) was thus established. IVL is a rare subtype of extranodal diffuse large B cell lymphoma with a poor outcome. However, it is recently thought that if the diagnosis is established early, aggressive chemotherapy increases survival. Senile angioma is a skin eruption that is considered prevalent for the most part in elderly people. If a patient is suspected to have IVL, and there is no appropriate site of biopsy, it might be beneficial to try a skin biopsy aiming at senile angiomas for early diagnosis.

A Model of Left Ventricular Dysfunction Complicated by CAWS Arteritis in DBA/2 Mice.

It was reported previously that a Candida albicans water-soluble fraction (CAWS), including a mannoprotein and ß-glucan complex, has strong potency in inducing fatal necrotizing arteritis in DBA/2 mice. In this study, histopathological changes and cardiac function were investigated in this system. One mg/day of CAWS was given to DBA/2 mice via peritoneal injection for five days. The CAWS-treated DBA/2 mice were induced aortitis and died at an incidence of 100% within several weeks. Histological findings included stenosis in the left ventricular outflow tract (LVOT) and severe inflammatory changes of the aortic valve with fibrinoid necrosis. Cardiomegaly was observed and heart weight increased 1.62 fold (P < 0.01). Echocardiography revealed a severe reduction in contractility and dilatation of the cavity in the left ventricle (LV): LV fractional shortening (LVFS) decreased from 71% to 38% (P < 0.01), and the LV end-diastolic diameter (LVDd) increased from 2.21 mm to 3.26 mm (P < 0.01). The titer of BNP mRNA increased in the CAWS-treated group. Severe inflammatory changes resulting from CAWS brought about lethal LV dysfunction by aortic valve deformation with LVOT stenosis. This system is proposed as an easy and useful experimental model of heart failure because CAWS arteritis can be induced by CAWS injection alone.

Severe pulmonary toxoplasmosis mimicking viral pneumonitis after a third allogeneic stem cell transplantation in a man with acute lymphoblastic leukemia.

A 22-year-old man with acute lymphoblastic leukemia underwent allogeneic stem cell transplantation (allo-SCT) twice, then underwent allo-SCT a third time due to relapse. On day 27, he developed acute respiratory distress, and bilateral interstitial infiltrates were noted on CT images. Despite receiving intensive treatment, the patient died on day 32 from progressive respiratory failure. An autopsy revealed evidence of diffuse alveolar damage caused by the genus Toxoplasma. At present, toxoplasmosis is considered to be a rare infectious complication in Japan. However, the actual incidence of toxoplasmosis may be higher than currently believed due to a lack of suspicion of the diagnosis in patients, difficulty in making a diagnosis and low autopsy rates.

Characterization of quasispecies of pandemic 2009 influenza A virus (A/H1N1/2009) by de novo sequencing using a next-generation DNA sequencer.

Pandemic 2009 influenza A virus (A/H1N1/2009) has emerged globally. In this study, we performed a comprehensive detection of potential pathogens by de novo sequencing using a next-generation DNA sequencer on total RNAs extracted from an autopsy lung of a patient who died of viral pneumonia with A/H1N1/2009. Among a total of 9.4x10(6) 40-mer short reads, more than 98% appeared to be human, while 0.85% were identified as A/H1N1/2009 (A/Nagano/RC1-L/2009(H1N1)). Suspected bacterial reads such as Streptococcus pneumoniae and other oral bacteria flora were very low at 0.005%, and a significant bacterial infection was not histologically observed. De novo assembly and read mapping analysis of A/Nagano/RC1-L/2009(H1N1) showed more than x200 coverage on average, and revealed nucleotide heterogeneity on hemagglutinin as quasispecies, specifically at two amino acids (Gly(172)Glu and Gly(239)Asn of HA) located on the Sa and Ca2 antigenic sites, respectively. Gly239 and Asn239 on antigenic site Ca2 appeared to be minor amino acids compared with the highly distributed Asp239 in H1N1 HAs. This study demonstrated that de novo sequencing can comprehensively detect pathogens, and such in-depth investigation facilitates the identification of influenza A viral heterogeneity. To better characterize the A/H1N1/2009 virus, unbiased comprehensive techniques will be indispensable for the primary investigations of emerging infectious diseases.

The first autopsy case of pandemic influenza (A/H1N1pdm) virus infection in Japan: detection of a high copy number of the virus in type II alveolar epithelial cells by pathological and virological examination.

We report the pathological and virological findings of the first autopsy case of the 2009 pandemic influenza (A/H1N1pdm) virus infection in Japan. A man aged 33 years with chronic heart failure due to dilated cardiomyopathy, mild diabetes mellitus, atopic dermatitis, bronchial asthma, and obesity died of respiratory failure and multiple organ dysfunction syndrome. Macroscopic examination showed severe pulmonary edema and microscopically the lung sections showed very early exudative-stage diffuse alveolar damage (DAD). Immunohistochemistry revealed proliferation of the influenza (A/H1N1pdm) virus in alveolar epithelial cells, some of which expressed SAalpha2-3Gal on the cell surface. Influenza (A/H1N1pdm) virus genomic RNA and mRNA were also detected in alveolar epithelial cells. Real-time PCR revealed 723 copies/cell in the left lower lung section from which the influenza (A/H1N1pdm) virus was isolated. Electron microscopic analysis revealed filamentous viral particles in the lung tissue. The concentrations of various cytokines/chemokines in the serum and the autopsied lung tissue were measured. IL-2R, IL-6, IL-8, IL-10, IFN-alpha, MCP-1, and MIG levels were elevated in both. These findings indicated a case of viral pneumonia caused by influenza (A/H1N1pdm) virus infection, showing characteristic pathological findings of the early stage of DAD.

Differential detection of PAS-positive inclusions formed by the Z, Siiyama, and Mmalton variants of alpha1-antitrypsin.

Several point mutations of alpha(1)-antitrypsin cause a perturbation in protein structure with consequent polymerization and intracellular accumulation. The retention of polymers of alpha(1)-antitrypsin within hepatocytes results in protein overload that in turn is associated with juvenile hepatitis, cirrhosis, and hepatocellular carcinoma. The detection of alpha(1)-antitrypsin polymers and understanding the molecular basis of polymer formation is of considerable clinical importance. We have used a monoclonal antibody (ATZ11) that specifically recognizes a conformation-dependent neoepitope on polymerized alpha(1)-antitrypsin to detect polymers within hepatocytes of individuals with alpha(1)-antitrypsin deficiency. Paraffin-embedded liver tissue specimens were obtained from individuals who were homozygous for the Z (Glu342Lys), Mmalton (52Phe del), and Siiyama (Ser53Phe) alleles of alpha(1)-antitrypsin that result in hepatic inclusions and profound plasma deficiency. Immunohistological staining with a polyclonal anti-human alpha(1)-antitrypsin antibody showed hepatic inclusions in all 3 cases, while ATZ11 reacted with hepatic inclusions formed by only Z alpha(1)-antitrypsin. Polymers of plasma M and Z alpha(1)-antitrypsin prepared under different conditions in vitro and polymers of recombinant mutants of alpha(1)-antitrypsin demonstrated that the monoclonal antibody detected a neoepitope on the polymerized protein. It did not detect polymers formed by a recombinant shutter domain mutant (that mirrors the effects of the Siiyama and Mmalton variants), polymers formed by cleaving alpha(1)-antitrypsin at the reactive loop, or C-sheet polymers formed by heating alpha(1)-antitrypsin in citrate. In conclusion, the ATZ11 monoclonal antibody detects Z alpha(1)-antitrypsin in hepatic inclusions by detecting a neoepitope that is specific to the polymeric conformer and that is localized close to residue 342.