Characterizing Adversity of Lysosomal Accumulation in Nonclinical Toxicity Studies: Results from the 5th ESTP International Expert Workshop.

Lysosomes have a central role in cellular catabolism, trafficking, and processing of foreign particles. Accumulation of endogenous and exogenous materials in lysosomes represents a common finding in nonclinical toxicity studies. Histologically, these accumulations often lack distinctive features indicative of lysosomal or cellular dysfunction, making it difficult to consistently interpret and assign adverse dose levels. To help address this issue, the European Society of Toxicologic Pathology organized a workshop where representative types of lysosomal accumulation induced by pharmaceuticals and environmental chemicals were presented and discussed. The expert working group agreed that the diversity of lysosomal accumulations requires a case-by-case weight-of-evidence approach and outlined several factors to consider in the adversity assessment, including location and type of cell affected, lysosomal contents, severity of the accumulation, and related pathological effects as evidence of cellular or organ dysfunction. Lysosomal accumulations associated with cytotoxicity, inflammation, or fibrosis were generally considered to be adverse, while those found in isolation (without morphologic or functional consequences) were not. Workshop examples highlighted the importance of thoroughly characterizing the biological context of lysosomal effects, including mechanistic data and functional in vitro readouts if available. The information provided here should facilitate greater consistency and transparency in the interpretation of lysosomal effects.

Landau renormalizations of superfluid density in the heavy-fermion superconductor CeCoIn5.

The formation of heavy-fermion bands can occur by means of the conversion of a periodic array of local moments into itinerant electrons via the Kondo effect and the huge consequent Fermi-liquid renormalizations. Leggett predicted for liquid (3)He that Fermi-liquid renormalizations change in the superconducting state, leading to a temperature dependence of the London penetration depth Λ quite different from that in BCS theory. Using Leggett's theory, as modified for heavy fermions, it is possible to extract from the measured temperature dependence of Λ in high quality samples both Landau parameters F(0)(s) and F(1)(s); this has never been accomplished before. A modification of the temperature dependence of the electronic specific heat C(el), related to that of Λ, is also expected. We have carefully determined the magnitude and temperature dependence of Λ in CeCoIn(5) by muon spin relaxation rate measurements to obtain F(0)(s) = 36 ± 1 and F(1)(s) = 1.2 ± 0.3, and we find a consistent change in the temperature dependence of C(el). This, the first determination of F(1)(s) with a value ≪ F(0)(s) in a heavy-fermion compound, tests the basic assumption of the theory of heavy fermions, that the frequency dependence of the self-energy is much more important than its momentum dependence.

Distribution of Taenia saginata metacestodes: a comparison of routine meat inspection and carcase dissection results in experimentally infected calves.

A comparison of techniques for detecting the presence of Cysticercus bovis in bovine carcasses was made by using carcass dissection and routine beef inspection guidelines. In the study, 28 calves were used after they were tested and found to be negative for the presence of anti-C. bovis serum antibodies and were inoculated orally with aliquots containing 6×10(4) Taenia saginata eggs. One hundred and twenty days after inoculation, the animals were slaughtered and a post mortem evaluation was done following Brazilian Federal Beef Inspection guidelines. This routine meat inspection was able to identify 71·42% of the assessed infected carcasses as being parasitized. This result implies that 28·58% of the infected carcasses would have been released as fit for human consumption since they would have been considered as free of C. bovis infection when using this method for carcass assessment. Only 3·07% of the total 2311 metacestodes present in the carcasses were identified by the conventional procedures of sanitary inspection. The assessment of different parts of the carcasses showed high infestation rates in shoulder clod (14·37%), head (11·21%), neck+chuck roll (8·05%), heart (7·75%) and top (inside) round (7·18%) which, together, were responsible for housing 48·51% of all the cysts found in the 24 beef cuts assessed. These numbers contrasted to the low incidence of cysts found in organs such as tongue (3·12%), diaphragm (1·69%) and esophagus (1·60%) which are usually described as predilection sites for the parasite.

Thoracic mass in a cynomolgus macaque (Macaca fascicularis).

A male cynomolgus macaque at the age of 3 years and 11 months suffered sudden cardiac arrest during a surgical operation. This animal had been clinically asymptomatic for 6 months from the acclimatization period to death. At necropsy, a white mass approximately 5 cm in diameter was found at the base of the heart. Histopathologically, the mass consisted of a granuloma with a number of multinucleated giant cells and multiple necrotic foci. Fungal hyphae characterized by parallel cell walls, distinct septa, and branching were observed in the lesion. The granuloma extended into the thoracic lymph nodes and the subepicardium of the left atrium, compressed the bronchioli, and was separated from the pulmonary parenchyma by a thick fibrous layer. The hyphal morphology and results of polymerase chain reaction assays demonstrated that the pathogen was Aspergillus sp.

Gel phase formation at resin-modified glass-ionomer/tooth interfaces.

Ionic bonding between polyalkenoic acid and hydroxyapatite may explain the excellent bonding retention of glass-ionomers in clinical trials. We have here investigated the extent to which the self-adhesiveness of resin-modified glass-ionomers (RMGIs) can be attributed to this chemical bonding capacity. Therefore, the interaction of 3 RMGIs with tooth substrates was comprehensively characterized, with electron and atomic force microscopy correlated with x-ray photoelectron spectroscopy (XPS). Interfacial ultrastructural analysis for 2 RMGIs disclosed a shallow hybridization of hydroxyapatite-coated collagen, on which a submicron gel phase was deposited through reaction of the polyalkenoic acid with calcium extracted from the dentin surface. One RMGI, however, bonded to dentin without hybrid layer or gel phase formation. XPS indicated that polycarboxylic acids included in the RMGIs electrostatically interacted with hydroxyapatite. We conclude that the self-adhesiveness of RMGIs should be attributed to ionic bonding to hydroxyapatite around collagen, and to micro-mechanical interlocking for those RMGIs that additionally hybridize dentin.

Assessing consumer expectations about pizza: A study on celiac and non-celiac individuals using the word association technique.

The word association (WA) technique was used to investigate the perception of two groups of consumers (72 celiac and 78 non-celiac individuals; 150 in total) to pizza dough (thick or thin) and the raw material used at the manufacture (cassava flour or rice flour). Different perceptions of the four stimuli were detected by Chi-square test (X2=314.393, p<0.0001) for both groups. Seven categories were used for both groups: food/composition, health, doubt/uncertainty, novelty, negative feelings, positive feelings, and sensory aspects. The stimulus 'pizza dough made with cassava flour' was associated with the category "food/composition" and the stimuli 'pizza made with rice flour', 'pizza made with cassava flour' and 'thin dough' were associated with "positive feelings". The stimulus 'thick dough' was related only to the category "negative feelings". WA indicated that gluten-free pizza should have thin dough and us cassava flour or rice flour as the raw material.

Geographical pathology of duck livers infected with duck hepatitis B virus from Chiba and Shimane in Japan and Shanghai in China.

In order to evaluate geographical differences in the liver pathology of ducks infected with duck hepatitis B virus (DHBV), ducks in Chiba and Shimane, Japan, and Shanghai, China, were investigated. The numbers (DHBV positive/negative) and the maximum age of the ducks examined were 18/10 at 19 mo, 15/1 at 3 yr 4 mo, and 72/27 at 18 mo, respectively. DHBV infection was induced experimentally in ducks from Chiba and Shimane but was present congenitally in those from Shanghai. Ducks were examined regarding liver function tests, conventional histology, immunohistology, electron microscopy, and molecular hybridization for DHBV DNA in the serum and liver. There was no significant difference between DHBV-positive and -negative ducks in bilirubin and transaminase and alkaline phosphatase activities in the sera. Histologically, while the livers of ducks from Chiba and Shimane did not show necroinflammatory (hepatitis) activity, those from Shanghai frequently did (52.5%). Necroinflammatory activity of the Shanghai ducks was present almost equally in both DHBV-positive and -negative livers. The livers of Shanghai ducks but not the other two areas often (8.3%) had ground-glass inclusions which corresponded ultrastructurally to numerous virus particles in the dilated cisternae of the proliferated endoplasmic reticulum. No advanced liver disease, such as cirrhosis or hepatocellular carcinoma, was observed. There was no significant difference in the amount of DHBV DNA in the sera or in its pattern in the liver tissue among ducks of the three areas. In addition, the livers of Chiba ducks frequently had amyloidosis, while those of Shanghai ducks were contaminated with parasites. In conclusion, DHBV infection did not appear to provoke significant hepatitis activity or advanced liver disease in the examined ducks of all three areas, and the DHBV-positive livers from Shanghai ducks showed a different morphological appearance from those of the other two areas. This variation might reflect the difference in the strain of ducks, subtypes of DHBV, environmental factors, or a combination of these influences.

Pericentric region of chromosome 9 is a possible candidate region for linkage study of schizophrenia.

We have undertaken a systematic G-banding survey to find structural chromosomal abnormalities among patients with schizophrenia. Of 120 patients with DSM-III-R schizophrenia, four (3.3%) had a pericentric inversion of chromosome 9 and three (2.5%) had a X/XX mosaicism. The frequency of pericentric inversion of chromosome 9 among patients with schizophrenia was statistically higher than those among newborns and Asian populations. Our results indicate that the pericentric region of chromosome 9 might be one of the potential regions of interest for linkage analysis of schizophrenia.

Changes of immunological functions after acute exacerbation in schizophrenia.

We investigated the changes of immunological functions in 14 schizophrenic patients (DSM-III-R; six men and eight women) who were hospitalized due to acute exacerbation of schizophrenia. The following immunological functions were studied on admission, 4 and 8 weeks after admission: serum immunoglobulins (Ig)G, A, and M; serum complement CH50; lymphocyte responses to mitogens (phytohemagglutinin, concanavalin A, and pokeweed mitogen); lymphocyte subpopulations (CD3%, 4%, 8%, 16%, 20%, 25%, and 56%); and natural killer cell (NK) activity. Psychological status of the patients, which was assessed by using Brief Psychiatric Rating Scale, improved gradually after admission. Changes in immune functions were analyzed using one-way analysis of variance and a randomized block analysis of variance with multiple comparison. NK activity on admission was significantly lower than those at 4 and 8 weeks after admission (p < .03). Serum IgG levels on admission and at 4 weeks after admission were significantly decreased as compared with those at 8 weeks after admission (p < .05); they were also lower than those in controls (p < .05). CD56% on admission and CD25% 4 weeks after admission were significantly increased as compared with controls (p < .05). These results indicate that several immunological functions might change related to time course after acute exacerbation. It is suggested that clinical conditions be carefully taken into consideration to evaluate immunological studies in schizophrenia.

No evidence of linkage or allelic association of schizophrenia with DNA markers at pericentric region of chromosome 9.

Based on our previous study suggesting the pericentric region of chromosome 9 as of potential importance in schizophrenia, we have carried out a linkage study between the schizophrenia phenotype and the dinucleotide repeat polymorphisms D9S55, D9S15, and D9S202 in three pedigrees multiply affected with schizophrenia. In addition, we have conducted allelic association studies using 60 patients with schizophrenia and 60 controls with polymorphisms at D9S55 and D9S15 markers. No evidence for linkage or association was found. The results indicate that susceptibility genes for schizophrenia are less likely to be located at the pericentric region of chromosome 9, assuming genetic homogeneity of the pedigrees.

Effects of diltiazem on plasma level and urinary excretion of digoxin in healthy subjects.

To evaluate a possible effect of diltiazem hydrochloride (DTZ) on digoxin (DX) kinetics, we performed a study in which a single oral dose of DX (0.5 or 0.75 mg) was given with and without DTZ (30 mg three times daily for 1 wk) to six healthy subjects. DTZ increased plasma DX concentrations at 3, 4, 6, and 12 hr and decreased renal clearance of DX from 3.05 +/- 0.126 to 2.31 +/- 0.234 ml/min/kg. There was no significant change in absorption t 1/2, peak concentration, peak concentration time, distribution t 1/2, biologic elimination t 1/2, or apparent volume of distribution with DTZ.

Schizophrenia and the HLA-DRB1 gene in the Japanese population.

OBJECTIVE: Small Japanese studies have suggested that patients with schizophrenia have higher rates of the HLA-DR1 gene than normal subjects. The authors' goal in the present study was to confirm this finding in a larger number of Japanese subjects. They also investigated the rate of DR4 in Japanese patients with schizophrenia because it has been reported that Caucasian patients with schizophrenia have higher rates of DR4. METHOD: They studied the occurrence of the HLA-DRB1 gene in 233 unrelated Japanese patients with schizophrenia compared with the occurrence of the gene in a group of 493 healthy Japanese volunteers. RESULTS: A larger proportion of the patients with schizophrenia (15.9%) than the comparison subjects (10.5%) were found to have DR1 (DRB1*0101). The proportion of patients (36.9%) and comparison subjects (40.6%) with DR4 did not differ significantly. CONCLUSIONS: Consistent with the findings of three other Japanese studies, the findings of the present study suggest that the rate of HLA-DR1 may be higher in Japanese patients with schizophrenia than in normal Japanese subjects. No evidence for an association between schizophrenia and the rate of DR4 was obtained in this study, although the combined data from the present study and other Japanese studies support the finding of lower rates of DR4 among patients with schizophrenia.

Structural abnormalities in frontal, temporal, and limbic regions and interconnecting white matter tracts in schizophrenic patients with prominent negative symptoms.

OBJECTIVE: Imaging studies of schizophrenia have repeatedly demonstrated global abnormalities of cerebral and ventricular volumes. However, pathological changes at more local levels of brain organization have not yet been so clearly characterized because of the few brain regions of interest heretofore included in morphometric analyses as well as heterogeneity of patient samples. METHOD: Dual echo magnetic resonance imaging (MRI) data were acquired at 1.5 T from 27 right-handed patients who met DSM-IV criteria for schizophrenia with enduring negative symptoms and from 27 healthy comparison subjects. Between-group differences in gray and white matter volume were estimated at each intracerebral voxel after registration of the images in standard space. The relationship between clinical symptom scores and brain structure was also examined within the patient group. Spatial statistics and permutation tests were used for inference. RESULTS: Significant deficits of gray matter volume in the patient group were found at three main locations: 1) the left superior temporal gyrus and insular cortex, 2) the left medial temporal lobe (including the parahippocampal gyrus and hippocampus), and 3) the anterior cingulate and medial frontal gyri. The volume of these three regions combined was 14% lower in the patients relative to the comparison subjects. White matter deficits were found in similar locations in the left temporal lobe and extended into the left frontal lobe. The patient group showed a relative excess of gray matter volume in the basal ganglia. Within the patient group, basal ganglia gray matter volume was positively correlated with positive symptom scores. CONCLUSIONS: Anatomical abnormalities in these schizophrenic patients with marked negative symptoms were most evident in left hemispheric neocortical and limbic regions and related white matter tracts. These data are compatible with models that depict schizophrenia as a supraregional disorder of multiple, distributed brain regions and the axonal connections between them.

Human leukocyte antigen and season of birth in Japanese patients with schizophrenia.

OBJECTIVE: Five Japanese studies, to the authors' knowledge, without exception, have consistently shown an increased frequency of human leukocyte antigen (HLA)-DR1 in patients with schizophrenia. This suggests an association between HLA-DR1 and schizophrenia in the Japanese population. The mechanism of the association is unknown; however, prenatal infections may be involved. The present study explored factors, including winter birth, that might correlate with this mechanism. Age at onset and gender were also studied. METHOD: Factors were compared between Japanese patients with schizophrenia with and in those without HLA-DR1 (N=60 and N=307, respectively). RESULTS: A significantly higher incidence of births in February and March was observed in patients with (31.7%) than those without (15. 6%) HLA-DR1. No association was found between the presence of HLA-DR1 and other variables. CONCLUSIONS: Although this result is preliminary, it may suggest an interaction between HLA and winter birth in the development of schizophrenia in the Japanese population.

Analysis of gastrin receptor gene expression in proliferating cells in the neck zone of gastric fundic glands using laser capture microdissection.

Gastrin stimulates proliferation of progenitor cells in the neck zone of gastric fundic mucosa. However, whether it directly enhances this proliferation through its receptors remains unclear. We investigated the expression of gastrin receptors in neck zone proliferating cells in rat gastric fundic glands using a reverse transcription polymerase chain reaction (RT-PCR) coupled with laser capture microdissection and in situ RT-PCR. Gastrin receptor expression was identified in c-fos-expressing cells located in the neck zone, and results of the RT-PCR analysis argued against contamination by other cells, such as enterochromaffin-like, parietal or D cells. Supporting this finding, gastrin receptor gene expression was identified in the neck zone as well as base glands by in situ RT-PCR. Therefore, it is suggested that proliferating cells in the neck zone are stimulated directly by gastrin via their gastrin receptors.

Direct determination of carbon and nitrogen contents of natural bacterial assemblages in marine environments

In order to better estimate bacterial biomass in marine environments, we developed a novel technique for direct measurement of carbon and nitrogen contents of natural bacterial assemblages. Bacterial cells were separated from phytoplankton and detritus with glass fiber and membrane filters (pore size, 0.8 &mgr;m) and then concentrated by tangential flow filtration. The concentrate was used for the determination of amounts of organic carbon and nitrogen by a high-temperature catalytic oxidation method, and after it was stained with 4',6-diamidino-2-phenylindole, cell abundance was determined by epifluorescence microscopy. We found that the average contents of carbon and nitrogen for oceanic bacterial assemblages were 12.4 +/- 6.3 and 2.1 +/- 1.1 fg cell-1 (mean +/- standard deviation; n = 6), respectively. Corresponding values for coastal bacterial assemblages were 30.2 +/- 12.3 fg of C cell-1 and 5.8 +/- 1.5 fg of N cell-1 (n = 5), significantly higher than those for oceanic bacteria (two-tailed Student's t test; P < 0.03). There was no significant difference (P > 0.2) in the bacterial C:N ratio (atom atom-1) between oceanic (6.8 +/- 1.2) and coastal (5.9 +/- 1.1) assemblages. Our estimates support the previous proposition that bacteria contribute substantially to total biomass in marine environments, but they also suggest that the use of a single conversion factor for diverse marine environments can lead to large errors in assessing the role of bacteria in food webs and biogeochemical cycles. The use of a factor, 20 fg of C cell-1, which has been widely adopted in recent studies may result in the overestimation (by as much as 330%) of bacterial biomass in open oceans and in the underestimation (by as much as 40%) of bacterial biomass in coastal environments.

Unusual relapse of adult T-cell leukemia/lymphoma after spontaneous remission.

A 42-year-old man was diagnosed with acute adult T-cell leukemia/lymphoma (ATL/L). Abnormal peripheral blood cells (45% of white blood cells) (Fig. 1a), hypercalcemia, and systemic lymphadenopathy were observed. Flow cytometric analysis (FCM) using peripheral mononuclear cells (PMNC) revealed that the immunophenotype of tumor cells was CD4+ CD8- CD25+ CD45RA- CD45RO+. Nevertheless, he developed a spontaneous remission 6 months later. At remission, the number of CD4-, CD25-, and CD45RO-positive cells decreased, while CD8- and CD45RA-positive cells increased to normal levels as previously reported by Suzuki et al. [1]. He was then referred to the outpatient clinic where he was periodically evaluated and received no therapy. Because of a serious sense of fullness he was re-admitted 30 months after diagnosis. Physical examination revealed ascites and small lymphadenopathy in the right axilla. Atypical lymphoid cells were not observed on microscopic examination of the blood smear. FCM using PMNC revealed that CD4+ CD25+ cells (3%) were within the normal range. Serum calcium was also within the normal range. Abdominal ultrasound examination showed massive ascites. Paracentesis demonstrated that the ascitic fluid had a high white blood cell count (3.15 x 10(9)/l) with a marked increase in abnormal large cells (Fig. 1b). FCM using mononuclear cells in the fluid revealed that 87.3% of the cells were double-positive for CD4 and CD25. Southern blot analysis of the cells confirmed monoclonal integration of human T-lymphotropic virus type 1 (HTLV-1) proviral DNA. The integrated genome was considered to be identical with that detected at initial presentation (Fig. 2). A diagnosis of relapsed ATL/L, with the same clone as was detected at initial diagnosis, was made. Although he was treated with cytotoxic drugs, he did not respond and he died of renal failure 1 month after relapse. Autopsy revealed nodular invasive lesions at the rectovesical pouch, omentum, diaphragm, and pericardium with peritoneal dissemination.

Clinical significance of LEA-1 expression in adult acute myeloid leukemia.

In this study, we examined expressions of several adhesion molecules (AdMs), i.e. leukocyte function antigen-1 (LFA-1: CD11a/CD18), Hermes homing receptor (CD44) and intercellular adhesion molecule-1 (ICAM-1: CD54), on leukemia cells from 51 adult patients with newly diagnosed acute myeloid leukemias (AMLs) to elucidate clinical significance of these AdM expressions. Those expressions in lymphoid malignancies have been correlated with tumor evolutions, but CD44 was detected in all the AML cases examined and CD54 expression did not associate with their clinical characteristics or outcomes. However, we found that LFA-1 expressions significantly correlated with splenomegaly, resistance to induction chemotherapies and short survival periods in AML patients.

Further evidence of no linkage between schizophrenia and the dopamine D3 receptor gene locus.

The dopamine hypothesis of schizophrenia proposed that dopaminergic pathways are involved in the etiology of the disease. In particular, interest among psychiatrists has focused on the D2 receptor because of its affinity to antipsychotic drugs. Recently a new dopamine receptor gene has been cloned, and named the dopamine D3 receptor. The D3 receptor is a potential site for antipsychotic drug action and may be involved in the pathophysiology of schizophrenia. We have carried out a linkage study between the susceptibility gene for schizophrenia and polymorphism of the dopamine D3 receptor gene in two Japanese pedigrees. The LOD scores were negative for all genetic models and for all affective status at a recombination fraction theta = 0. Linkage of DRD3 has been excluded for the model 1 (dominant model) and the model 3 (recessive model). The LOD score was -3.43 at theta = 0 for model 1 (dominant model) and broad definition of affected status. These results were consistent with previous studies.