Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.     

Adenosquamous carcinoma of the gall-bladder with gastric foveolar-type epithelium

An 80 year old Japanese man had adenosquamous carcinoma of the gall-bladder characterized by an adenocarci-noma (AC) in the gall-bladder lumen and a squamous cell carcinoma (SCC) in the Invaded region of the liver. In the AC, the tumor cells consisted of atypical columnar epithelium with pseudostratification, mimicking gastric foveolar epithelium, while atypical signet-ting cells were scattered within the SCC. There was an abrupt transition between the AC and SCC areas. The tumor cells in the AC area were intensely positive for galactose oxidase-Schiff staining, and paradoxical concanavalin A staining revealed these tumor cells to have Class II mucins. lmmunohistochemically, the tumor cells in foveolar-type adenocarcinoma were diffusely positive for cathepsin D. Flow cytometrical analysis of DNA content showed the AC area to be diploid and the SCC area to be aneuploid. The Sphase fraction of the SCC area (46.9%) was larger than that of the AC area (19.5%). The positive rate of immunostaining for proliferating cell nuclear antigen in the SCC area (mean 50.627%) was larger than that of the AC area (mean 3.048%, P < 0.01). These resutts suggest that the AC area of this tumor, histochemically and immunohistochemically, showed gastric foveolar-type characteristics, the SCC component was squamous cell metaplasia of the preexisting AC, and that the SCC area had a greater proliferating capacity than the AC area.     

Agarose for a bioartificial pancreas.

Islets were encapsulated into 5% concentration agarose microbeads. The effect of microencapsulation on islet allograft survivals was determined using a streptozotocin-induced diabetic (STZ) mouse and a nonobese diabetic (NOD) mouse as recipients. All five STZ BALB/c mice receiving microencapsulated islets (C57BL/6) maintained normoglycemia indefinitely. When NOD mice were used as recipients of the bioartificial pancreas, four of five grafts (islets from C3H/He) functioned for more than 80 d. Two of five NOD mice maintained normoglycemia until animals were sacrificed at 102 and 192 postoperative d. Microbeads made of commercially available agarose can effectively prolong alloislets functioning in the STZ-diabetic mouse and even in the NOD mouse (animal model of human type I diabetes) without the use of any immunosuppressive drug.     

TCR repertoire in early fetal mouse thymus

We investigated the rearrangement and expression of TCR genesin mouse fetal thymus organ culture, a system that avoids subsequententry of hematopoietic precursor cells. The first observablerearranged TCR gene was homogeneous V2-J2, detectable as earlyas fetal day 11 (d11) in the thymic primordla. The productiveTCR was homogeneous V5-J1, first detectable in d13 thymocytes,followed by adult-type TCR (V4 and V7). Sequence analysis ofTCR revealed five types of V-J junctional sequences. In thevery early stage, a homogeneous V-J junction is generated viaa short homology sequence in the coding region (Type I), whilea short homology sequence in the P-nucleotlde rather than thecoding region is used in the following stage (Type II). In thelater embryonic stages, diverse V-J junctions are generatedby well-known mechanisms, such as P-nucleotide (Type III), N-regioninsertion (Type IV) or trimming of the coding ends (Type V).These findings suggest that the generation of homogeneous TCR (V2 and V5) in the early fetal stages is due to the intrinsicrearrangement mechanisms and is in stage specific manner.     

Granulocytic sarcoma presenting with lymph node infarction at disease onset

A completely infarcted lymph node is an unusual event. However, lymph node infarction should alert the pathologist to the considerable likelihood of malignant lymphoma. We report two unusual cases of acute myeloid leukemia presenting with granulocytic sarcoma at disease onset with a lymph node lesion exhibiting extensive lymph node infarction. The infarcted tissue contained numerous eosinophilic cell ghosts. There were some islands of degenerated, pyknotic medium-sized nuclei resembling lymphoblasts present in the necrotic area. By immunohistochemistry, these medium sized cells were CD3-, CD20-, CD34+, CD43+, CD45RO-, CD68-, CD79a- and myeloperoxidase+ in both cases. Differentiation of granulocytic sarcoma from malignant lymphomas is important for adequate therapy. The present cases indicate that granulocytic sarcoma should be added to the list of differential diagnoses for lymph node infarction.     

Immunoregulatory defects of V alpha 24V+ beta 11+ NKT cells in development of Wegener's granulomatosis and relapsing polychondritis

The frequency of either CD4(-)8(-) (double negative; DN) or CD4(+) V alpha 24(+)V beta 11(+) NKT cells, the expression of CD1d and the binding of CD1d-tetramer loaded with alpha-galactosylceramide (alpha-GalCer) to NKT cells were analysed in peripheral blood mononuclear cells (PBMCs) of patients with Wegener's granulomatosis (WG), relapsing polychondritis (RP) and healthy subjects (HS). DN and CD4(+) V alpha 24(+)V beta 11(+) NKT cells as well as CD1d-alpha-GalCer tetramer-positive NKT cells, were significantly decreased in number in both WG and RP patients compared to those from HS. When cytokine profiles were analysed in these PBMCs upon stimulation with phorbol ester and calcium ionophore, CD4(+) T cells from patients with WG and RP exhibited a Th1 bias, whereas CD4(+) NKT cells from WG patients in remission showed a Th2 bias. These findings suggest that NKT cells (especially CD4(+) NKT cells) play a regulatory role in Th1 autoimmunity in patients with WG and RP. The reduction in NKT cell counts appears to be associated with the low responsiveness to alpha-GalCer. The dysfunction of NKT cells to recognize ligands such as alpha-GalCer may also contribute to the defects observed in NKT cells from WG and RP patients.     

Immunohistochemical analysis of centromere protein F expression in buccal and gingival squamous cell carcinoma

Centromere protein F (CENP-F) expression (localization and characteristics) in relation to tumor clinicopathological parameters was immunohistochemically examined and evaluated in 47 archival biopsy specimens of buccal and gingival squamous cell carcinomas (SCC). Centromere protein F expression was detected in 79% of the samples. An increase in the labeling index (LI) with WHO grading was obtained ( P  < 0.05). Correlations were obtained between the CENP-F LI and tumor size ( P  < 0.05). Immunoelectron microscopy showed CENP-F nuclear staining as punctate or fine dots. The present study shows that CENP-F expression and detection of a more specific cell subpopulation presents a theoretical advantage for the analysis of the precise cell cycle of G₂ to M cells, compared to Ki-67.     

Identification and characterization of PDZRN3 and PDZRN4 genes in silico

NUMB and NUMBL are implicated in cell fate determination through the inhibition of Notch signaling. LNX, binding to NUMB and CXADR (CAR), functions as E3 ubiquitin ligase at least for NUMB. LNX is the paralog of PDZRN1 (PDZ domain containing RING finger 1). Here, we identified two novel homologs of LNX and PDZRN1 by using bioinformatics, which were designated PDZRN3 (LNX3 or SEMCAP3) and PDZRN4 (LNX4 or SAMCAP3L), respectively. KIAA1095 cDNA (AB029018) was the representative PDZRN3 cDNA. Complete coding sequence of PDZRN4 cDNA was determined by assembling nucleotide sequences of ESTs (BF059062 and AW297403), FLJ33777 cDNA (AK091096) and IMAGE5767589 cDNA (BC040922). PDZRN4 gene, consisting of 11 exons, was found to encode two isoforms with N-terminal divergence (PDZRN4 and PDZRN4S) due to an alternative promoter. PDZRN3-CNTN3 locus at human chromosome 3p13-p12.3 and PDZRN4-CNTN1 locus at human chromosome 12q12 were paralogous regions within the human genome. PDZRN3 (1066 aa) and PDZRN4 (1036 aa) showed 59.9% total-amino-acid identity. Two bipartite nuclear localization signals (NLS) were located within the C-terminal region of PDZRN3 and PDZRN4. PR34H1 and PR34H2 domains were identified as the regions conserved among PDZRN3, PDZRN4 and Drosophila CG1783. PDZRN3 and PDZRN4 consist of RING, two PDZ, PR34H1, PR34H2 domains and two NLS, while PDZRN1 and LNX consist of RING and four PDZ domains. PDZRN family proteins were classified into the LNX-PDZRN1 subfamily and the PDZRN3-PDZRN4 subfamily. This is the first report on the PDZRN3 and PDZRN4 genes.     

Primary pulmonary low-grade marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue type with prominent hyalinosis. A case report

We report a case of primary pulmonary low-grade marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT)-type with prominent sclerosis, which morphologically resembled pulmonary hyalinizing granuloma (PHG) or inflammatory pseudotumor (IPT) of the lung. The patient, a 66-year-old Japanese female with a history of Sj?gren's syndrome and primary biliary cirrhosis, presented with a lower left lobe mass 6.8 cm in diameter. Histologically, the lesion is characterized by dense bundles of collagen with scattered plasma cells, mature small lymphocytes, and histiocytes among the collagen bundles. Only the peripheral area of the nodule contained dense lymphoplasmacytoid and histiocytoid infiltrates. A few centrocyte-like cells were obscured by the numerous plasma cells and plasmacytoid cells. In addition, lymphoepithelial lesions and colonalized lymphoid follicles were identified by immunohistochemistry alone. Although PHG and IPT are unlikely to be confused with pulmonary MALT-type lymphomas, the present case suggests that MALT-type lymphoma should be added to the list of differential diagnoses for PHG and IPT.