Cell proliferation marker MCM2, but not Ki67, is helpful for distinguishing between minimally invasive follicular carcinoma and follicular adenoma of the thyroid.

AIMS: To compare cell proliferation markers, minichromosome maintenance protein 2 (MCM2) and Ki67, in minimally invasive follicular carcinoma (MIFC) and follicular adenoma (FA) of the thyroid and among MIFCs with different diagnostic criteria. METHODS AND RESULTS: Twenty-two MIFCs and 20 FAs were immunohistochemically stained for MCM2 and Ki67. The MIFCs were subdivided into six Group 1 tumours with both capsular and vascular invasions, seven Group 2 tumours with vascular invasion only and nine Group 3 tumours with capsular invasion only. The MCM2 and Ki67 indices were calculated, counting more than 1000 tumour cells in the most frequently positive areas. In total and Groups 1-3 MIFCs and in FAs, the average MCM2 index was 26.7 +/- 11.0, 28.4 +/- 8.6, 26.3 +/- 14.8, 25.9 +/- 8.4 and 10.7 +/- 4.5, respectively, whereas the average Ki67 index was 2.07 +/- 1.65, 1.93 +/- 2.02, 2.49 +/-1.38, 1.84 +/- 1.5 and 1.78 +/- 0.92, respectively. There was a significant difference in the MCM2 index, but not in the Ki67 index, between each category of MIFCs and FA (P < 0.01). However, neither the MCM2 index nor the Ki67 index showed a statistically significant difference among the subgroups of MIFC. CONCLUSIONS: MCM2, but not Ki67, is a helpful marker for differentiating MIFC from FA. The tumour cell proliferative activity supports the histological criteria based on diagnosing MIFC by either capsular or vascular invasion only.

Acute interstitial pneumonitis during chemotherapy for haematological malignancy.

Fourteen adult patients with haematological malignancies (eight non-Hodgkin's lymphoma, one multiple myeloma, one chronic lymphocytic leukaemia, two acute lymphoblastic leukaemia and two acute myeloid leukaemia) developed acute interstitial pneumonitis (IP) during the course of chemotherapy. All patients manifested high fever over 38 degrees C, bilateral diffuse pulmonary interstitial infiltrates in the chest radiograph and severe hypoxia without hypercapnia in the arterial blood gas analysis. Pathogenic microorganisms were not detected in repeated examinations in any patient. Chemotherapy given included various anti-neoplastic drugs. Five patients had received granulocyte colony-stimulating factor (G-CSF) for chemotherapy-induced leucopenia. The onset was associated with an increase of leucocytes in 10 patients. All patients were treated with high dose steroid hormone and broad spectrum antibiotics with or without anti-fungal agents, and three required mechanical ventilation. Eleven patients quickly recovered from these situations, whereas three died. Autopsies were done in two patients and disclosed pneumocystis carinii (PC) pneumonitis in one and non-specific pulmonary congestive oedema and fibrosis in the other. In conclusion, IP of unknown cause could develop in patients with various haematological malignancies especially at the recovery phase of chemotherapy-induced leucopenia irrespective of the previous G-CSF administration. High dose steroid hormone should be used as therapy for such patients as soon as possible after exclusion of an infective aetiology.

An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells.

Specialized DNA sequences known as insulators protect genes from both the positive and negative influences of nearby chromatin. Many insulators have been identified in various species; however, few function in multiple species. We have shown that an insulator from the Ars (arylsulfatase) gene of the sea urchin Hemicentrotus pulcherrimus functions in plant cells. Normally, expression of an introduced chimeric GUS gene is inactivated in approximately 30% of transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. The insulator did not affect the copy number, the chromosomal position of transgene integration or maximum expression levels. These results suggest that the insulator functions to suppress the variation normally associated with transgene expression in tobacco BY2 cells.

Isolation of growth-phase-specific promoters from cultured tobacco cells.

Strong promoters are required under several culture conditions for effective transgene expression in tobacco BY2 cells. We have isolated the promoter fragments of 4 genes exhibiting high homology to those of Arabidopsis thaliana 108C1T7 (unknown function) and F1-ATPase-delta, alcohol dehydrogenase and pectin esterase genes from a genomic DNA library of BY2 cells. Two of the four genes were strongly expressed during every phase of growth of BY2 cells, and the other two were expressed only during the stationary phase. Each of the promoter fragments was ligated to the GUS reporter gene and introduced into the chromosome of BY2 cells by Agrobacterium-mediated transformation. Growth-phase-dependent expression of the GUS gene was reproduced under the control of all 4 promoters observed with the original genes. Significantly higher expression was observed under the control of Nt108p during every phase of cell growth and under the control of NtADHp and NtPESp during the stationary phase than that under the control of the CaMV35S promoter.

Metabolic engineering of cultured tobacco cells.

Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor. Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5'-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells. Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture.

Possible involvement of protein phosphatase type 2A in tumor necrosis factor alpha-induced tissue factor expression in endothelial cells.

The involvement of protein Ser/Thr phosphatase types 2A (PP2A) and 1 (PP1) in tumor necrosis factor alpha (TNF alpha)-induced tissue factor (TF) expression of human umbilical vein endothelial cells (HUVEC) was investigated. PP2A was more abundant than PP1 in the cytosol of HUVEC. CAL-A (0.5 nM) and OKA (2 nM), cell permeable inhibitors of PP1 and PP2A, augmented TNF alpha-induced TF expression, although TF expression induced by either TPA or thrombin was unchanged in the presence of GAL-A. Addition of CAL-A (0.5 nM) to TNF alpha-stimulated cultures led to an increase in the accumulation of TF transcripts. CAL-A (0.5 nM) also augmented the TNF alpha-induced phosphorylation of I kappa B-alpha, an inhibitor of NF-kappa B. Since PP2A is more sensitive to OKA than PP1, these results suggest that PP2A may be involved in regulating TNF alpha-induced TF expression in HUVEC through I kappa B-alpha dephosphorylation.

Plasma levels of activated FVII in various diseases.

Plasma activated factor VIIa (FVIIa) levels were measured in various diseases using mutant tissue factor (TF). FVIIa levels in thrombotic patients and patients with idiopathic thrombocytopenic purpura were significantly higher than those in healthy control subjects. The plasma FVIIa levels in thrombotic patients treated with warfarin were similar to those of control subjects. The plasma FVIIa levels in pregnant women and patients with systemic lupus erythematosus, infection or malignancies were high. However, the levels in patients with disseminated intravascular coagulation (DIC) were not significantly increased. DIC patients are in a severe hypercoagulable state, and exhibit severe consumption of coagulation factors. The slightly increased FVIIa level in the DIC patients observed is probably considered to be caused by consumption of coagulation factors. The plasma FVIIa level was poorly correlated with other hemostatic parameters except for protein C in our analysis of all cases. In the analysis of DIC and thrombotic patients treated without warfarin, the plasma FVIIa level was negatively correlated with TF antigen. Plasma FVIIa levels might reflect hypercoagulability in thrombotic diseases, and a normalized FVIIa level in patients with thrombotic diseases should be considered to be associated with DIC.

Gastric syphilis: polymerase chain reaction detection of treponemal DNA in pseudolymphomatous lesions.

Syphilis is an unexpected diagnosis in the stomach. To establish the diagnosis, evidence of Treponema pallidum in the gastric lesion is necessary. However, it is sometimes difficult to prove the presence of the organisms by conventional methods. The authors describe two cases of early gastric syphilis with pseudolymphomatous histology in which T pallidum gene was detected by the polymerase chain reaction (PCR) using paraffin biopsy sections. The gastric lesion of each case endoscopically and histologically simulated that of malignant lymphoma. However, no clonality was proved by immunohistochemistry or PCR gene rearrangement analysis. No spirochetal organisms were detected with certainty by Warthin-Starry silver stain, whereas the organisms were shown by immunofluorescent stain in one patient. A PCR study showed the treponemal DNA in both patients, and its validity was supported by a direct sequencing and a restriction enzyme digestion. Positive results of serological tests for syphilis and regression of the lesions after antisyphilitic treatment were confirmatory of the diagnosis. Gastric syphilis should be considered as a differential diagnosis when an atypical lymphoid infiltrate fails to show monoclonality. The present PCR method would be helpful in showing T pallidum using routinely processed small biopsy specimens as the tissue source.

Increased plasma-soluble fibrin monomer levels in patients with disseminated intravascular coagulation.

Plasma-soluble fibrin monomer (SFM) level in patients with disseminated intravascular coagulation (DIC) was significantly higher than the level in patients with pre-DIC or in non-DIC patients, and the level in patients with pre-DIC was significantly higher than that in non-DIC patients. There was no significant difference in plasma SFM levels among various diseases underlying DIC. Plasma SFM level in patients with good outcome was significantly decreased after treatment for DIC. The sensitivity of fibrin degradation products and platelet number was high for DIC, but not for pre-DIC. The sensitivity of thrombin-antithrombin III complex, plasmin-plasmin inhibitor complex, and SFM was high for both DIC and pre-DIC. The specificity of these markers was also high. Receiver operating characteristic analysis suggests that plasma SFM level could be the most useful marker for the diagnosis of both DIC and pre-DIC.

[Plasma fibrin monitor level in DIC patients with a hematopoietic malignancy].

The Plasma level of soluble fibrin monomer (sFM) was measured in 218 patients with a hematopoietic malignancy. Of them, 198 were diagnosed with disseminates intravascular coagulation (DIC), 20 with Pre-DIC, and 20 with Non-DIC. Pre-DIC was retrospectively defined as the condition at least 1 week before the onset of DIC. The plasma levels of sFM, thrombin-anti-thrombin III complex (TAT), plasmin alpha 2-antiplasmin inhibitor complex (PIC), and FDP-D-dimer were significantly higher in patients with DIC than in those with Non-DIC. These levels were significantly higher in patients with Pre-DIC than in those with Non-DIC. Among these hemostatic parameters, the plasma sFM showed the highest sensitivity and specificity for DIC or Pre-DIC. These findings suggests that sFM is the most valuable marker hemostatic for the diagnosis of DIC and Pre-DIC.

Hemostatic abnormalities and increased vascular endothelial cell markers in patients with red cell fragmentation syndrome induced by mitomycin C.

We examined red cell fragmentation syndrome (RCFS) induced by mitomycin C (MMC) (13 patients), by thrombotic thrombocytopenic purpura (TTP) (17 patients), and by disseminated intravascular coagulation (DIC) (15 patients). Plasma cytokine levels were increased in the TTP and DIC patients, but not in those whose RCFS was induced by MMC, suggesting that the activation of the immune system plays an important role in the pathogenesis of RCFS due to TTP and DIC but did not in RCFS due to MMC. Plasma thrombomodulin, tissue type plasminogen activator, and plasminogen activator inhibitor-I levels were increased in all RCFS patients, suggesting that RCFS, whether MMC induced, or due to TTP or DIC, might be associated with vascular endothelial cell injury. In TTP, von Willebrand factor (vWF) antigen and high molecular weight vWF multimer levels were reduced, possibly as a result of microthrombus consumption. The hemostatic data in this study showed that the TTP patients were in a hypercoagulable state without hyperfibrinolysis, and that DIC patients were in both a hypercoagulable and a hyperfibrinolytic state, whereas hemostatic abnormalities were slight in patients with MMC induced RCFS. These findings suggest that vascular endothelial cell injuries might be associated with RCFS, and that those injuries in MMC-induced RCFS might not be related to microthrombi or an activated immune system.

Outcome of disseminated intravascular coagulation in relation to the score when treatment was begun. Mie DIC Study Group.

We examined 395 patients with disseminated intravascular coagulation (DIC) divided into two groups: non-leukemic and leukemic. In 58% of the patients as a whole, treatment of DIC resulted in complete or partial remission, while exacerbation and death occurred in 31%. The efficacy of DIC treatment in the non-leukemic group was less than that in the leukemic group, indicating that the outcome of DIC depended, in part, on the underlying disease. We examined hemostatic indicators in relation to DIC score: prothrombin time (PT) ratio, FDP, platelet count, and fibrinogen levels were found to be important indicators for the diagnosis of DIC, but not for Pre-DIC. Plasma levels of fibrin-D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PPIC) were significantly increased in pre-DIC. The efficacy of treatment in relation to the DIC score when the treatment was begun showed that greater efficacy was achieved in pre-DIC than in DIC patients. The outcome was poorer with increasing DIC score, suggesting that early diagnosis and early treatment are important. On examining the relationship between outcome and hemostatic indicators, we found that the PT ratio and the levels of antithrombin, plasminogen, PPIC, the PPIC/TAT ratio, and thrombomodulin were related to outcome, suggesting that very high consumption of blood coagulation factors, liver dysfunction, hypofibrinolysis, or organ failure caused a poor outcome. Although the outcome in DIC patients may not depend substantially on plasma levels of TAT and fibrin-D-dimer, we can use these indicators to treat DIC patients at an early stage.

[Acute promyelocytic leukemia following ulcerative colitis].

We report a case of acute promyelocytic leukemia (APL) following ulcerative colitis (UC). A 23-year-old man was diagnosed as UC in January 1991 and had been treated with salazosulfapyridine and prednisolone with good effect. In September 1993, he developed bleeding tendency and a diagnosis of APL with disseminated intravascular coagulation was made based on the results of bone marrow aspiration and coagulation profile. Complete remission was achieved with All-trans retinoic acid together with combined chemotherapy. He died of sepsis during consolidation chemotherapy in December 1993. Autopsy revealed no recurrence of UC.

Monoclonality in gastric lymphoma detected in formalin-fixed, paraffin-embedded endoscopic biopsy specimens using immunohistochemistry, in situ hybridization, and polymerase chain reaction.

Diagnosis of gastric malignant lymphoma remains a challenge, especially when the tissue source is endoscopic biopsy specimens. Once an atypical lymphoid infiltrate is found, demonstration of clonality is the key to establishing a diagnosis of the disease. For this purpose, we evaluated the usefulness of immunohistochemistry, in situ hybridization, and polymerase chain reaction (PCR) using formalin-fixed, paraffin-embedded endoscopic materials from 20 patients with B-cell malignant lymphomas. Template DNA for PCR was obtained by microdissecting Giemsa-stained sections using a serial hematoxylin and eosin section as a guide. Clonal rearrangement bands were demonstrated in 15 of 20 cases (75%) by PCR, whereas expression of monotypic light-chain mRNA was detected in seven of 20 (35%) by in situ hybridization and monotypic light-chain restriction in four of 20 (20%) by conventional immunohistochemistry. Although less sensitive than PCR, in situ hybridization was useful for localizing the expression of target mRNAs with cellular accuracy and with low background staining. In addition, two cases were found to be monoclonal only by in situ hybridization, and not by PCR. The results showed that clonal proliferation is detected with the greatest sensitivity with PCR using small routinely processed biopsy specimens and that a difficulty with the PCR method in terms of cellular localization was partially overcome using a microdissection procedure that provided at least tissue-level accuracy.

Effect of lipoproteins on tissue factor activity and PAI-II antigen in human monocytes and macrophages.

Expression of the tissue factor (TF) and the plasminogen activator inhibitor-II were induced in cultured human monocytes-macrophages by incubation with lipoproteins. Very low-density lipoprotein (VLDL) augmented the TF and PAI-II expression the most, followed by low-density lipoprotein (LDL) and a very weak effect by high-density lipoprotein (HDL). In macrophages pre-cultured for 3 days, oxidized LDL augmented the expression of TF activity in the macrophages to a greater extent than native LDL. These findings indicate that lipoproteins affect both monocytes and macrophages, and that they induce a hypercoagulable-hypofibrinolytic state. Thus hyperlipidemia may be a direct risk factor for thrombotic disease.

Quadruple cancers in a human T-cell leukemia virus type 1 carrier.

Human T-cell leukemia virus type 1 (HTLV-1) infection is considered to contribute to the risk of malignancies other than adult T-cell leukemia. We report a 64-year-old male HTLV-1 carrier who developed quadruple malignancies such as cancer of the urinary bladder, skin, larynx and liver.