A case of methotrexate-related lymphoproliferative disease showing multiple liver lesions in a patient with rheumatoid arthritis.

A 66-year-old woman with rheumatoid arthritis (RA) who had been receiving methotrexate (MTX) for 2 years presented with tarry stools. Contrast-enhanced computed tomography (CT) of the abdomen revealed irregular wall thickening in the ileocecal region and multiple low-contrast masses in both lobes of the liver. Lower gastrointestinal endoscopy revealed a type 2 tumor in the ileocecal region with a semi-peripheral ulcer. Histological examination of liver and colon biopsies showed other iatrogenic immunodeficiency-associated lymphoproliferative disorder (Oi-LPD), diffuse large B-cell lymphoma type, with positivity for Epstein-Barr virus DNA. After withdrawal of MTX, the LPD lesions disappeared and the patient achieved remission. We considered this to be a sporadic case of Oi-LPD, diffuse large B-cell lymphoma type, in the liver and colon due to treatment with MTX. There has been no previous report of this condition with simultaneous hepatic and colonic lesions, and the present case is thought to be highly informative in relation to the pathogenesis.

Postmortem diagnosis of fatal anaphylaxis during intravenous administration of therapeutic and diagnostic agents: evaluation of clinical laboratory parameters and immunohistochemistry in three cases.

This report documented three autopsy cases involving fatal shock during intravenous injection of therapeutic and diagnostic agents in a hospital setting. For postmortem diagnosis, clinical laboratory parameters for anaphylaxis, specificity of antibodies for allergens and mast cell numbers in tissue sections were examined. Elevated plasma tryptase levels were evident in the three adult males; two of the three victims displayed elevated IgE levels. However, immunoassay failed to detect antibodies specific to the relevant agent. Double immuno-staining was performed employing anti-tryptase and anti-chymase monoclonal antibodies in order to count mast cells in lung sections. Increased numbers of mast cells were observed in anaphylactic tissues, which was particularly true for chymase-positive cells, in comparison with tissues associated with acute traumatic deaths. In addition to findings at autopsy, positive data obtained by laboratory examinations and immunohistochemical analyses indicated that fatal systemic anaphylaxis occurred during intravenous injection of clinical agents.

Sensitive detection of polyalanine expansions in PHOX2B by polymerase chain reaction using bisulfite-converted DNA.

Congenital central hypoventilation syndrome, also known as Ondine's curse, is characterized by idiopathic abnormal control of respiration during sleep. Recent studies indicate that a polyalanine expansion of PHOX2B is relevant to the pathogenesis of this disorder. However, it is difficult to detect the repeated tract because its high GC content inhibits conventional polymerase chain reaction (PCR) amplification. Here, we describe a bisulfite treatment for DNA in which uracil is obtained by deamination of unmethylated cytosine residues. Deamination of DNA permitted direct PCR amplification that yielded a product of 123 bp for the common 20-residue repetitive tract with replacement of C with T by sequencing. It settled allele dropouts accompanied by insufficient amplification of expanded alleles. The defined procedure dramatically improved detection of expansions to 9 of 10 congenital central hypoventilation syndrome patients examined in a previous study. The chemical conversion of DNA before PCR amplification facilitates effective detection of GC-rich polyalanine tracts.

Usefulness of serum mast cell-specific chymase levels for postmortem diagnosis of anaphylaxis.

Chymase, a serine protease, is stored mainly in secretory granules of human mast cells. Serum chymase concentration was examined in 8 autopsy cases with anaphylaxis as well as in 104 control cases without anaphylaxis. It was detected in all 8 cases with anaphylaxis (range 3-380 ng/ml, mean 89.8 ng/ml), while it was detected in only 2 of the 104 controls and was below a detectable level (<3 ng/ml) in the other 102. Serum tryptase levels are known to be a diagnostic indicator of anaphylaxis, therefore the relationship between serum chymase and tryptase levels was investigated in the 8 cases of anaphylactic death; a significant positive correlation was found (r=0.826, p=0.011). Furthermore, chymase was shown to be quite stable in serum. These results showed that measurement of serum chymase levels might be an additional tool for postmortem diagnosis of anaphylaxis.

Association of alpha2-HS glycoprotein (AHSG, fetuin-A) polymorphism with AHSG and phosphate serum levels.

Alpha2-HS glycoprotein (AHSG), also known as fetuin-A, is a plasma protein displaying high-affinity interaction with calcium phosphate, by which ectopic vascular calcification is prevented. This investigation has attempted to evaluate the relationship between AHSG polymorphism and serum levels of AHSG and calcium-related parameters. AHSG levels in unrelated individuals were measured by quantitative rocket immunoelectrophoresis and were 581+/-38, 542+/-31, and 494+/-23 mg/l for three major genotypes of AHSG1 homozygotes (n=99), heterozygotes (n=55), and AHSG2 homozygotes (n=22), respectively (differences were significant: P<0.001). The circulating AHSG level was therefore influenced by the genetic polymorphism with the additive reduction in the AHSG2 allele. Statistical analysis of simple and multiple regression models revealed no associations between AHSG levels and serum values of total calcium, albumin-corrected total calcium, and ionized calcium. However, the AHSG levels demonstrated a significant negative correlation with free phosphate levels (P<0.001), indicating that AHSG is a novel determinant of serum phosphate. The AHSG polymorphism is attributable to the hereditary variation of AHSG and phosphate serum levels, which may affect skeletal development and chronic disorders such as vascular calcification.

Expression of the prolactin-inducible protein (PIP/GCDFP15) gene in benign epithelium and adenocarcinoma of the prostate.

Prolactin-inducible protein (PIP), also known as gross cystic disease fluid protein 15, is a predominant secretory protein in various body fluids, including saliva, milk and seminal plasma. Immunohistochemistry of this protein has been exploited as a clinical marker for breast cancer and Paget's disease. This study comparatively examined PIP expression in normal prostate tissues and in adenocarcinomas of the prostate. Quantitative real-time RT-PCR revealed low-level presence (6%) of PIP mRNA in normal prostate tissue in comparison with the seminal vesicle. Indirect immunostaining with monoclonal antibody 3E7 displayed a positive sign for benign epithelium in 8 cases (29.6%) among 27 normal specimens; however, the incidence significantly increased to 56.1% (37/66) in instances involving primary prostate carcinoma tissues of different types. Quantitative RT-PCR also demonstrated that PIP transcript levels in carcinoma regions were significantly higher than corresponding levels in benign regions. These findings conclusively showed that benign prostate epithelium expresses PIP at low levels; in contrast, PIP is over-expressed in carcinomas of the prostate.

Divergent evolution of the prolactin-inducible protein gene and related genes in the mouse genome.

The prolactin-inducible protein (PIP/GCDFP15) family consists of small secretory polypeptides that are found in various body fluids. In order to study evolutionary events to this family, we cloned member genes and analyzed their sequences. A database search revealed the presence of a novel paralogous gene on mouse chromosome 6q34 and a nonprocessed pseudogene adjacent to PIP on human chromosome 7q34. The mouse PIP and four related genes displayed higher nonsynonymous and synonymous substitution ratios in comparison to other mammalian PIP orthologues; furthermore, these genes exhibited distinct distributions among tissues such as seminal vesicle, colon, and mammary gland. A pair of duplicated genes could have existed prior to radiation to the human and rodents. While only PIP is active in the human lineage, species-specific gene duplications have given rise to functional variants in rodents. Adaptive evolution potentially has occurred among the PIP and its related genes in the mouse genome.

A death in a stationary vehicle whilst idling: unusual carbon monoxide poisoning by exhaust gases.

In this paper, we describe an autopsy case in which death was due to accidental carbon monoxide poisoning occurring in a stationary vehicle idling in an open space. To investigate the source of the fatal fumes, the death scene situation was reconstructed using the vehicle. Exhaust gases were found to invade the interior through the floor from a defective exhaust system. CO gas was detected while idling and the level in the cabin gradually rose to 1.5% over a 2-h period. Since the 8-year-old motor vehicle seemed to have been defective for some months, it was concluded that stationary idling overnight caused an accumulation of toxic gases in the interior.

Molecular characterization of a human monoclonal antibody to B antigen in ABO blood type.

A human anti-B antibody of clone BT97 was obtained from a healthy individual of type A of the ABO blood group without immunization. Cloning was performed by means of heterohybridoma formation of cell fusion between human peripheral lymphocytes and mouse myeloma cells. The antibody selectively reacted with B-antigen in flow cytometry using red blood cells and enzyme-linked immunosorbent assay. The VH and VL genes of BT97 were derived from the germline genes of DP-47 and 3p.81A4, respectively, with a couple of somatic mutational events. Comparative analysis with other reported anti-A, B and H antibodies revealed that the amino acid sequence of the VH region was more homologous than that of the VL region. The sequence of BT97 showed complete identity with one anti-H natural antibody reported by Marks et al., with the exception of the CDR3 region. It is not known whether the homologies include the common properties of the natural antibodies; however, a particular germline gene potentially changes to anti-ABH antibodies. We think that this method is suitable for cDNA preparation of human monoclonal antibodies to blood group antigens and for sequence analysis.

Evolution of the cystatin B gene: implications for the origin of its variable dodecamer tandem repeat in humans.

The human cystatin B gene contains a variable number of 12-bp tandem repeats in its promoter region, of which the common alleles contain two or three copies and unusual expansion causes progressive myoclonus epilepsy of the Unverricht-Lundborg type. We undertook a comprehensive analysis of the genomic sequence to address the evolutionary events of this variable repeat. By examination of a contiguous genome sequence spanning 5.0 kb and linkage analysis of detected polymorphic changes, we identified six major intragenic haplotypes in unrelated Japanese subjects. The number of normal repeats was closely correlated with these alleles, indicating that changes in the array should be comparatively rare events during human evolution. To examine the origin of the repeat array further, we also analyzed five primate genomes. Repetitive polymorphism was unlikely in hominoids, and the array originated with the dodecamer itself in the course of primate evolution. The variability conceivably developed after the separation to humans.