The application of molecular techniques to solid tumors

Recent developments in the field of molecular biology including the sequencing of the human genome and related high throughput methodologies are presenting the diagnostic pathologist with new opportunities to expand our understanding of human disease. These techniques enable the comprehensive assessment of molecular alterations with cell populations of interest, including cancer. It will be necessary for the diagnostic pathologist to become familiar with these techniques to effectively translate their potential into the clinical environment.     

The tyrosine-based YXXØ targeting motif of murine leukemia virus envelope glycoprotein affects pathogenesis

Retroviruses, such as human and simian immunodeficiency viruses (HIV and SIV), and murine leukemia viruses (MuLV), harbor a tyrosine-based motif in the intracytoplasmic domain of their envelope glycoprotein. This motif can act as an endocytosis signal or as a targeting signal, restricting viral budding at specific cell surface membrane domains. In the present study, proviral DNA of the ecotropic Cas-Br-E strain of MuLV was modified by substitution or deletion of the critical tyrosine residue. Mutant viruses lost basolateral targeting in polarized MDCK epithelial cells while expression level of the glycoprotein at the cell surface was not affected. This suggests that the tyrosine-based motif in MuLV does not act as an endocytosis signal. Only a small delay in the appearance of disease was observed in inoculated mice. In contrast, a striking change in the pathology was observed with enlarged thymus and lymph nodes in animals inoculated with mutant viruses.     

Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection

Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.     

Alveolar type II cell abnormalities and peroxide formation in lungs of rats given IL-1 intratracheally

Acute lung injury (ALI) is characterized by increased lung levels of proinflammatory cytokines, inflammation, oxidative stress, edema, and impaired gas exchange. Notably, ALI patients also exhibit pulmonary surfactant abnormalities, including increased levels of phospholipids in their lung lavages. In the present study, to assess early alterations of the lung surfactant system in ALI, we induced inflammation and acute lung injury in rats by administering interleukin-1 (IL-1) intratracheally. Five h after IL-1 instillation, we examined lung tissue ultrastructure by electron microscopy using both routine staining methods and cerium chloride staining to localize hydrogen peroxide (H₂O₂) histologically. We also measured lung lavage phospholipid levels, lung tissue -glutamyl transpeptidase (GGT) activities (a marker of oxidative stress), and arterial blood oxygen tensions. We observed that lungs of rats given IL-1 intratracheally had increased neutrophil accumulation, increased H₂O₂ production, and increased alveolar type II (ATII) pneumocyte ultrastructural abnormalities compared to rats given saline intratracheally. Intratracheal instillation of IL-1 also increased phospholipid levels in the bronchoalveolar lavage (BAL), possibly as a consequence of the abnormal discharge of lamellar bodies into the alveolar lumen. In addition, IL-1-insufflated rats had increased lung GGT levels and impaired blood oxygenation compared to saline-insufflated rats. Treatment with mepacrine decreased lung neutrophil accumulation, ultrastructural lung abnormalities, lung lavage phospholipid levels, lung tissue GGT levels, and blood oxygenation impairment in rats given IL-1 intratracheally, suggesting a possible relationship between these events. Our results indicate that IL-1-induced acute lung injury in rats is marked by neutrophil-dependent oxidative stress, ATII cell defects, abnormal discharge of lamellar body phospholipids, and impaired blood oxygenation.     

Relationship between normal heart size and body indices in Korean

We provided a curve-fit equation to predict the normal heart weight (g) in Koreans by examining 422 autopsies (215 males and 207 females, from newborn to age 77 yr) who were relatively in good general condition. Heart weight was well correlated with body surface area (m2), body weight (kg), and body height (cm) but poorly with age in both sex. Heart weight progressively increased from birth to the earlier 3rd and 4th decades in male and female, respectively, and then gradually decreased; mean heart weight of all age group was greater in male than in female and significantly different from birth to 4th decade. In both sex, heart weight exponentially increased in accordance with the increase of body height, body weight, and body surface (in male, heart weight=0.00312 x body height(2.239), r2=0.750, p<0.0001; in female, heart weight=0.00443 x body height(2170), r2=0.781, p<0.0001; in male, heart weight=9.22 x body weight(0.853), r2=0.770, p<0.0001; in female, heart weight=9.00 x body weight0.855, r2=0.820, p<0.0001; in male, heart weight=155.18 x body surface area1.290, r=0.808, p<0.0001; in female, heart weight=124.13 x body surface area1.242, r=0.834, p<0.0001). These results indicate that heart weight is better correlated with body surface area than with body weight; however, body weight should be a better determinant of a predicted heart weight, since body surface area is entirely dependent on body height and body weight.     

Mutational analysis of Fas ligand gene in human non-Hodgkin lymphoma

Among the systems triggering apoptosis, the Fas-Fas ligand (FasL) system is recognized as a major pathway for the induction of apoptosis in cells and tissues. Ligation of Fas by either an agonistic antibody or FasL transmits a 'death signal' to the target cell, potentially triggering apoptosis. Alterations of genes along the Fas-mediated apoptosis pathway have been reported in many human cancers. However, there have been no data regarding FasL gene mutations in human cancers. We hypothesized that FasL gene mutation might be involved in the development of non-Hodgkin lymphoma (NHL). In this study, we analyzed the entire coding region of the FasL gene for the detection of somatic mutations in a series of 111 NHLs and found that one tumor had a FasL gene mutation in the cytoplasmic domain. To evaluate the functional alterations of the mutant in apoptosis, we overexpressed the mutant in 293T cells, but couldn't find any significant loss of cell death compared to the wild-type FasL. Together, these data suggest that FasL is occasionally mutated in human NHL and that FasL mutations appear to play no role in the pathogenesis of the vast majority of NHLs.     

Comparison of deltaFVC between patients with allergic rhinitis with airway hypersensitivity and patients with mild asthma.

BACKGROUND: In asthmatic individuals, airway sensitivity and maximal airway response are increased. Airway sensitivity is usually evaluated by measuring the provocation concentration of inhaled methacholine or histamine that causes a decrease in forced expiratory volume in 1 second of 20% (PC20). The percentage decrease in forced vital capacity at the PC20 (deltaFVC) has been proposed as a surrogate marker for maximal airway response. Individuals with allergic rhinitis and no clinical evidence of asthma frequently exhibit airway hypersensitivity. OBJECTIVE: To compare the deltaFVC between patients with allergic rhinitis and mild asthmatic patients with a similar degree of airway hypersensitivity. METHODS: A retrospective analysis of methacholine challenge test data from 72 children with allergic rhinitis and airway hypersensitivity (methacholine PC20 < 16 mg/mL) (rhinitis group) and from 72 children with mild atopic asthma matched to the rhinitis group regarding the methacholine PC20 (asthma group). The deltaFVC was calculated on the concentration-response curve to methacholine. RESULTS: The mean +/- SD deltaFVC was significantly lower in the rhinitis group (15.0% +/- 3.6%) vs the asthma group (17.4% +/- 5.3%) (P = .002). There was no significant correlation between the deltaFVC and PC20 in the rhinitis (r = -0.101; P = .41) and asthma (r = -0.023; P = .85) groups when 2 patients with PC20 less than 1 mg/mL were excluded from each group. CONCLUSIONS: Patients with allergic rhinitis and airway hypersensitivity had a significantly lower deltaFVC than methacholine PC20-matched mild asthmatic patients, suggesting that the level of maximal airway response in patients with allergic rhinitis is lower than that in mild asthmatic patients with a similar degree of airway hypersensitivity.     

Negative conversion of antimitochondrial antibody in primary biliary cirrhosis: a case of autoimmune cholangitis

Autoimmune cholangitis is a clinical constellation of chronic cholestasis, histological changes of chronic nonsuppurative cholangitis and the presence of autoantibodies other than antimitochondrial antibody (AMA). It is uncertain whether this entity is definitely different from AMA positive primary biliary cirrhosis (PBC), though it shows some differences. We report a case of autoimmune cholangitis in a 59-year-old woman, who had been previously diagnosed as AMA-positive PBC associated with rheumatoid arthritis, has been converted to an AMA-negative and anticentromere antibody-positive PBC during follow-up. The response to ursodeoxycholic acid treatment is poor except within the first few months, but prednisolone was dropping the biochemical laboratory data.     

Autoimmune haemolytic anaemias. V. Studies on the resistance against complement haemolysis of the red cells of patients with chronic cold agglutinin disease

Experiments are described the results of which sustain the hypothesis that resistance against complement haemolysis, which is a characteristic of the red cells of patients with chronic cold agglutinin disease, is due to the following mechanism: when red cells react with cold auto-agglutinins in vivo, they are either haemolysed immediately, or, due to an unknown factor, escape direct haemolysis. In the latter case β₁E and β₁A disappear from the cell membrane. To the sites where these proteins have been attached once, no new β₁E or β₁A molecules can be bound. Full complement activation thus becomes impossible.