Rat peripheral mononuclear cell thymidine kinase activity increases during liver regenerative processes after partial hepatectomy

Deoxythymidine kinase (TK; EC 2.7.1.21) activity in the liver has been used as a marker of liver regeneration after partial hepatectomy. In this study we examined TK activity of various organs, plasma and peripheral blood mononuclear cells (PMNC) in 70% partially hepatectomized rats. TK activity of lymph nodes, small intestine, heart, lung, kidney and thymus did not increase significantly during the course of the study, except for spleen at 72 h. On the other hand, PMNC-TK and liver cystosolic TK activity increased in a parallel fashion at all times after partial hepatectomy; they began to increase 12 h after surgery and peaked 48 h post-surgery. Fractionation of PMNC into T cells and B cells revealed that both populations increased and peaked 48 h post-surgery. Plasma TK peaked 12-24 h after surgery, then declined at 36, 48 and 72 h after partial hepatectomy. This change paralleled plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). PMNC-TK activity correlated significantly with liver cystosolic TK activity 24 h (r = 0.743; P < 0.05) and 48 h (r = 0.708; P < 0.05) after partial hepatectomy. However, it did not correlate with plasma levels of TK, AST and ALT. The results indicate that in the early stage of liver regeneration PMNC-TK may provide a marker of liver regenerative processes.     

Schistosomiasis around Siavonga, on the shores of Lake Kariba, Zambia

The DNA profile of blast cells was assayed in 61 children with acute leukemias (51 patients) and non-Hodgkins lymphomas (NHL--10 patients). The value of S phase (synthesis of DNA) and G2M phase (mitotic stage) was compared between the subtypes of acute leukemia and lymphoma based on blast cell phenotype. In acute lymphoblastic leukemia (ALL) the lowest S phase of blast cells was seen in null-ALL subtype, the highest in T-ALL. In non-lymphoblastic leukemia (ANLL) the value of S phase was below S phase observed in ALL. B cell NHL showed higher S phase as compared to T-lymphocyte derived NHL cells. Aneuploidy was noted as hyperdiploidy (8 cases), hypodiploidy (4 cases) and two leukemia cell lines (3 ALL patients). The DNA profile as marker of proliferative activity of blastic cells provides an important information associated with the prognosis of patient.     

Evaluation of maternal plasma creatine kinase activity as a marker of abnormal early pregnancy

We have tested the value of maternal plasma creatine kinase activity for diagnosing ectopic pregnancies obtained after in-vitro fertilization and embryo transfer. Plasma creatine kinase was assayed in 57 patients: 20 normal, 23 miscarriages and 14 ectopic pregnancies, for a total of 240 samples. All values were in the lower part of the normal range except only one in a miscarrying patient. A statistically significant difference was observed for a cut-off value of 45 IU/l between normal and ectopic pregnancies. However, for this cut-off point, the measurement of plasma creatine kinase activity had a sensitivity of 0.50 and a specificity of 0.76 for the diagnosis of ectopic pregnancy. The positive predictive value was 0.69. Creatine kinase activity measurements are thus of no practical value in this particular population, in which an early and specific marker of ectopic implantation would be of paramount interest. The association of human chorionic gonadotrophin (HCG) determinations and ultrasound scanning of the pelvis still remain the best paraclinical support for an early diagnosis of ectopic implantation.     

Inhibitory activity in human marrow plasma directed against mouse marrow cell proliferation: reduced in subjects with decreased granulopoiesis

Human venous plasma is known to contain a lipoprotein-inhibitor of mouse marrow cell growth which we have found does not have cell type-specificity, in that it inhibits mouse lymphoma cell as well as marrow cell colony formation in vitro. Following its removal by CHCl3, we have identified residual inhibitory activity which reduces the growth of mouse marrow cells but not lymphoma cells. This inhibitory activity was found to be present in marrow and to much lesser extent in peripheral venous plasma obtained from subjects without disturbances in granulopoiesis. It was markedly reduced in subjects with disorders in which normal granulopoiesis was reduced, such as acute leukemia. The deficiency of this inhibitory activity in the marrow plasma of subjects with leukemia and related disorders may be due to a reduction in the cells from which it is derived (e.g. normal neutrophils or stromal cells), although further studies will be required to verify its presence and to determine its source and physiological role in granulopoiesis in man.     

Total serum sialic acid is a general disease marker rather than a specific tumour marker in dogs

The aim of the present study was to examine the levels of total sialic acid (TSA) in serum of clinically healthy dogs and dogs with various diseases to evaluate the usefulness of TSA as a tumour marker. TSA levels in clinically healthy dogs were not different between sexes, but pregnant and lactating dogs had higher mean (+/- standard deviation (SD)) TSA levels than clinically healthy female dogs (642 +/- 78 vs. 495 +/- 73 mg/l, P < 0.001). Eighty-eight dogs with different tumours (54 malignant and 34 benign tumours of different tissues) had higher mean TSA levels than 148 clinically healthy dogs (675 +/- 143 vs. 498 +/- 75 mg/l, P < 0.01). Fifty dogs with other diseases excluding tumours (skin, urinary system, and gastrointestinal diseases, pyometra, other inflammatory diseases, and Cushing's syndrome) had slightly higher TSA levels than the tumour-bearing dogs (730 +/- 159 mg/l, P = 0.02). TSA levels in dogs with malignant tumours did not differ from dogs with benign tumours (682 +/- 144 vs. 664 +/- 142 mg/l, P = 0.73). A receiver-operating characteristic (ROC) plot revealed a maximum sensitivity and specificity combination of 69% and 91% (TSA cut-off concentration 595 mg/l) in distinguishing between healthy dogs and dogs with tumours. When evaluating TSA measurements to distinguish dogs with other diseases from dogs with tumours, a maximum sensitivity and specificity combination of 50% and 75% was found (cut-off concentration 761 mg/l). WHO staging of mammary tumours revealed an increase in TSA levels with increasing stage (P < 0.0001, rs, = 0.62). In conclusion, the nonspecificity of increases makes TSA determinations unsuitable as a tumour marker. TSA levels seem instead to be a general disease marker. Whether serial TSA measurements could be used in the follow-up of dogs operated for malignant tumours should be further investigated.     

Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines

In this study, we examined a large number of patients to clarify the distribution and frequency of a recently described FLT3 tandem duplication among hematopoietic malignancies, including 112 acute myelocytic leukemia (AML), 55 acute lymphoblastic leukemia (ALL), 37 myelodysplastic syndrome (MDS), 20 chronic myelogenous leukemia (CML), 30 non-Hodgkin's lymphoma (NHL), 14 adult T cell leukemia, 15 chronic lymphocytic leukemia (CLL) and 38 multiple myeloma (MM). We also evaluated 71 cell lines derived from 11 AML, 31 ALL, two hairy cell leukemia, three acute unclassified leukemia, 10 CML, 12 NHL including six Burkitt's lymphoma, and two MM. Using genomic PCR of exon 11 coding for the juxtamembrane (JM) domain and first amino acids of the 5'-tyrosine kinase (TK) domain, this length mutation was found only in AML (22/112, 20%) and MDS (1/37). According to the FAB subclassification, they were 5/18 (28%) of M1, 4/29 (14%) of M2, 3/17 (18%) of M3, 6/24 (25%) of M4, 4/20 (20%) of M5 and 1/9 of refractory anemia with excess of blast in transformation. In the various cell lines examined, this abnormality was determined in only one derived from AML and never found in other hematological malignancies. The sequence analysis of the abnormal PCR products revealed that 23 of 24 showed internal tandem duplication with or without insertion of nucleotides. In one AML, insertion and deletion without duplication was determined. All 24 lengthened sequences were in-frame. Duplication takes place in the sequence coding for the JM domain and leaves the TK domain intact. In conclusion, we emphasize that the length mutation of FLT3 at JM/TK-I domains were restricted to AML and MDS. Since all these mutations resulted in in-frame, this abnormality might function for the proliferation of leukemic cells.     

A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase

Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli as a thrombin cleavable fusion protein. The TK was expressed as an inducible glutathione S-acetyl transferase fusion protein and purified in a first step by glutathione affinity chromatography. Proteolytic cleavage of the column bound TK with thrombin led to a truncated enzyme, resulting from two new and hitherto unknown cleavage sites, determined by N-terminal sequencing. In a second step, the TK was further purified from the cleavage products by ATP affinity chromatography, yielding homogeneously pure TK as shown by SDS-PAGE and mass spectrometry. Both the fusion protein and the purified enzyme show enzymatic activity with the same Km value of 0.2 microM for the natural substrate thymidine. Determination of the native molecular weight indicated that the pure enzyme and the fusion protein are biologically active as homodimers. Therefore the recombinant enzyme has the same biochemical characteristics as the viral TK, expressed in infected cells.     

The thymidine kinase/ganciclovir-mediated "suicide" effect is variable in different tumor cells

The herpes simplex virus thymidine kinase (HSV-TK) converts ganciclovir (GCV) into a toxic product and allows selective elimination of TK+ cells in vitro and in vivo. It is currently being used in clinical gene therapy trials as a therapeutic gene or as a safety marker. We have analyzed the susceptibility of different tumor cell lines to the TK/GCV-mediated "suicide" effect. Therefore, tumor cells TSA, J558L, EB, and ESB and, as a control, NIH-3T3 cells were infected with a retrovirus containing a hygromycin/TK fusion gene. All cell lines were sensitive to GCV in vitro; however, the concentration of GCV and the time needed to eliminate tumor cells completely considerably varied between different tumor cell lines. TSA-TK cells were completely eliminated within 10 days in 1 microg/ml GCV, whereas ESB-TK cells required 22 days in 10 microg/ml GCV. When two cell lines were examined, the differing sensitivity to GCV in vitro correlated with the ability to eradicate TK+ tumors in vivo. TSA-TK tumors could be eliminated in almost all animals by systemic GCV administration, whereas ESB-TK tumors were completely resistant. Different sensitivity to GCV was not due to different TK expression levels because the cells were similarly resistant to hygromycin, and Western blot analysis with an anti-TK antiserum revealed similar protein amounts in TSA/TK and ESB-TK cells. Together, the results demonstrate that tumor cells are highly different concerning the susceptibility to the TK/GCV effect, which, however, may be tested for in vitro.     

Semi-quantitative analysis of telomerase in pancreatic ductal adenocarcinoma

Using a polymerase chain reaction-based amplification assay, we measured telomerase activity in surgically resected pancreatic ductal carcinomas (n = 16 cases) and normal ducts (n = 6), comparing findings with the telomerase activity of a human pancreatic cancer cell line, MIA PaCa-2, as a standard, i.e., relative telomerase activity was determined. Telomerase activity was expressed as the equivalent telomerase intensity of the number of cells of MIA PaCa-2 per microgram protein of tissue samples. The median value for telomerase activity in normal pancreatic ducts was 0.13 and the 25th and 75th percentile were 0.01 and 0.76. The median value for telomerase activity in pancreatic ductal adenocarcinoma was 34.7 (25th percentile, 4.98; and 75th percentile, 296), significantly higher than that of normal ducts (P < 0.001). When the cut-off value was set at 1.0 and 3.0, the telomerase positivity rate of pancreatic ductal adenocarcinomas was 100% and 81.3%, respectively. Telomerase may be specific marker for pancreatic ductal carcinomas.     

Diabetic gastroparesis]

OBJECTIVES: This study was undertaken to determine the clinical usefulness of nuclear matrix protein 22 (NMP22) as a novel urine marker for urothelial cancer, particularly, to substitute for voided-urine cytology. METHODS: NMP22 values were determined for 280 patients and 20 healthy volunteers by NMP22 Test Kit based on an enzyme-linked immunosorbent assay. RESULTS: When the cut-off value was set at 10 U/ml, the positive rate of urinary NMP22 for urothelial cancer was 80.9% (38/47), whereas that for posttreatment cases and benign diseases was 35.7% (74/207). When urinary NMP22 and voided-urine cytology were compared, the test for urinary NMP22 showed higher sensitivity than cytology in patients with urothelial cancer. When urinary NMP22 values were determined pre- and postoperatively in patients with urothelial cancer, the postoperative value decreased in all patients, and were below the cut-off value in all except one patient. CONCLUSIONS: Urinary NMP22 is a useful diagnostic marker as a substitute for voided-urine cytology for the surveillance of urothelial cancer.     

Intrahepatocellular erythrocyte inclusions and increased calcium precipitation in canine endotoxic shock

AIMS: To investigate the electron microscopic localization of membrane-bound and exchangeable calcium with specific calcium precipitation techniques during endotoxic shock in the dog. METHODS: Ten pentobarbital anesthetized, mechanically ventilated, and paralyzed dogs were studied. Six dogs received 2 mg/kg E. coli endotoxin i.v. followed by a continuous 0.9% saline infusion to restore and maintain baseline cardiac filling pressures. Four dogs served as time-matched controls. Each experiment lasted for 3 h. After the completion of study, the livers of four endotoxic and two control dogs were fixed by perfusion of 3% glutaraldehyde via the portal vein. Liver sections were then prepared for electron microscopy and calcium localization studies. RESULTS: Hepatocytes of endotoxic animals completely lost their plasma membrane-bound calcium. The most severely damaged cells showed extensive "blebbing" of the plasma membrane and contained numerous cytoplasmic erythrocyte inclusions. Endotoxin administration also caused excessive calcium precipitation inside hepatocytes in areas with pronounced sinusoidal damage. CONCLUSIONS: In this acute model of fluid-resuscitated endotoxic shock in dogs, the use of specific calcium localization techniques enables the demonstration of disturbances in hepatocellular calcium handling, which appear to be closely related to structural alterations of the hepatocyte cell membrane. Erythrocyte uptake by hepatocytes is a previously undescribed phenomenon in canine endotoxic shock and may serve as an additional histologic marker of ultrastructural cell (membrane) damage.     

Toward a final design for the Birmingham boron neutron capture therapy neutron beam

CD10 (CALLA) antigen is expressed in a wide variety of epithelial and nonepithelial tissues, but its most significant application is in the diagnosis and classification of certain types of malignant lymphoma and leukemia. CD10 is expressed in a high percentage of cases of acute lymphoblastic leukemia (ALL), follicular lymphoma, Burkitt's lymphoma, and some hematopoietic tumors. Although the antigen is not lineage specific, CD10 expression is widely used to define subgroups within B-ALL and is a useful tool for detecting the presence of leukemic blasts in the bloodstream. Currently available monoclonal antibodies to CD10 have been found to be effective only in fresh-frozen tissue and for techniques such as flow cytometry. We have used a recombinant protein corresponding to the whole of CD10 to generate a monoclonal antibody that is effective in paraffin-embedded tissue sections. We have used this antibody to assay for the presence of CD10 on a range of normal and pathological tissues. Strong staining was seen in lymphoid germinal centers, renal tubules, glomeruli, syncytiotrophoblast, hepatic parenchymal canaliculi, B-lineage ALL, follicle center cell lymphoma, and a proportion of cases of large-B-cell lymphoma. We believe that this antibody will be of value in the characterization of malignant lymphoma, in particular the differential diagnosis of small-B-cell lymphoma and subtyping of lymphoblastic leukemia, as well as the investigation of the significance of expression of CD10 in other normal and pathological tissues.     

The levels of granulocyte colony-stimulating factor in the plasma of the bone marrow aspirate in various hematological disorders

We developed a sensitive method of measurement of granulocyte colony-stimulating factor (G-CSF) by an enzyme-linked immunosorbent assay, which we applied in the plasma of the bone marrow aspirate in 70 patients with various hematological disorders. The lowest limit of detection by this method is 2 pg/ml. G-CSF was detected in all but two of the patients. Compared to the G-CSF level in normal healthy controls, those in non-Hodgkin's malignant lymphoma, aplastic anemia, agranulocytosis and multiple myeloma were significantly higher, while the level in refractory anemia was not different. The G-CSF level in acute myelogenous leukemia patients was either elevated or decreased regardless of the French-American-British subgroup. The level in acute lymphoblastic leukemia was not different from the normal value, as was that in refractory anemia with an excess of blasts, and that in chronic lymphocytic leukemia. A patient with chronic myelomonocytic leukemia showed initial elevation of G-CSF with normalization after entering complete remission. The G-CSF level in chronic myelogenous leukemia was significantly decreased, although one patient in hematological remission who was under alpha-interferon therapy showed normal levels. The level in polycythemia vera was not significantly different from the normal value. The G-CSF level for the entire group showed an inverse, although not statistically significant, correlation with the percentages of myeloid cells of the bone marrow (r = -0.174, p = 0.1703, n = 80). These results are thought to reflect the regulatory mechanism of granulopoiesis in the bone marrow in various hematological disorders, and it is concluded that this method may be of clinical use in the treatment of patients with these disorders and in the selection of candidates likely to benefit from G-CSF administration.     

Evaluation of an assay for detecting telomerase activity in neoplastic tissues of dogs

OBJECTIVE: To determine feasibility of using the telomere repeat amplification protocol (TRAP) assay to detect telomerase activity in tumors of dogs. SAMPLE POPULATION: Samples of tumor or normal tissue were obtained from client-owned dogs that underwent surgical biopsy during the period of January 1996 through December 1997. PROCEDURE: The TRAP assay was used to detect telomerase activity in malignant or benign tumors of dogs. Telomerase status (positive or negative) was compared with results of histologic examination for each sample to estimate specificity and sensitivity of this assay for the diagnosis of malignancy. RESULTS: Of 26 malignant tumors, 24 were telomerase positive on TRAP assay, whereas 3 of 4 benign tumors and 3 of 3 normal tissues were telomerase negative. Analysis of these results indicated an estimated sensitivity of 92% and specificity of 86% for tumor analysis, using the TRAP assay. CONCLUSION: The TRAP assay can be used to measure telomerase activity in malignant tumors of dogs. CLINICAL RELEVANCE: Because telomerase activation may be required for indefinite longevity of cells, it may also serve as a tumor marker and therapeutic target. The TRAP assay can be used to detect telomerase in samples of fluid as well as tissues obtained from solid tumors. Therefore, it may have considerable clinical value in rapid and noninvasive diagnosis of neoplasia in dogs. Additional studies must be completed to more accurately determine sensitivity and specificity of the assay.     

Renin-specific antibody for study of cardiovascular homeostasis

Antiserum specific for purified canine renal renin was used to inhibit this enzyme in trained, conscious dogs. The antiserum did not affect blood pressure in sodium-replete dogs but decreased plasma renin activity and blood pressure in sodium-depleted animals. The antiserum also reduced blood pressure to control levels concomitant with suppression of plasma renin activity in uninephrectomized dogs with acute renovascular hypertension. These observations establish the role of the renin-angiotensin system in the maintenance of blood pressure in the sodium-depleted state as well as in the initiation of renovascular hypertension.     

Long-term alterations in growth factor mRNA expression following seizures

Pneumocystis carinii pneumonia (PCP) is one of the most predominant opportunistic infectious diseases in patients with AIDS. Nested PCR has been described as a sensitive and specific tool for detecting P. carinii DNA in clinical specimens. Little is known about the correlation of positive PCR results and clinical evidence of PCP in patients with different forms of immunosuppression. One hundred and thirty-six sputum samples, 26 tracheal-bronchial aspirate samples, 35 bronchoalveolar lavage samples, and 11 lung biopsy samples from (i) human immunodeficiency virus (HIV)-infected patients with AIDS, (ii) immunocompromised patients with leukemia or lymphoma, and (iii) immunocompetent control patients were investigated by a nested PCR amplifying DNA from the mitochondrial large subunit of P. carinii. All patients suffered from acute episodes of respiratory disease. The resulting data were correlated with clinical evidence of PCP. A high degree of association of positive P. carinii PCR results and clinical evidence of PCP in HIV-infected patients with AIDS was found. When calculated for bronchoalveolar lavage and lung biopsy samples, the positive and the negative predictive values of P. carinii PCR for PCP diagnosis in HIV-infected patients with AIDS were 1 and the specificity and the sensitivity were 100%. In contrast, in the group of patients with leukemia or lymphoma, the positive predictive value of the nested PCR for these materials was found to be as low as 0.09, the negative predictive value was 0.73, the specificity was 44.4%, and the sensitivity was 25.0%. No P. carinii DNA could be detected in specimens from immunocompetent patients. In summary, in contrast to patients with leukemia and lymphoma, nested PCR seems to be a sensitive and specific tool for PCP diagnosis in HIV-infected patients with AIDS.     

白血病和淋巴瘤患者ABO血型分布及地区性差异分析

目的 探讨ABO血型与白血病、淋巴瘤的相关性和地区性差异.方法 采用病例对照研究方法,调查不同类型白血病、淋巴瘤患者和健康对照组ABO血型分布特征,分析不同地区白血病、淋巴瘤患者的ABO血型分布情况.结果 急性非淋巴细胞白血病、急性淋巴细胞白血病、非霍奇金淋巴瘤患者的ABO血型分布与健康人群分布差异有统计学意义(χ2=21.23、χ2=8.36、χ2=9.39,均P<0.05).国内不同地区的白血病、淋巴瘤ABO血型分布有差异,其中白血病ABO血型分布差异具有统计学意义(χ2=50.65,P<0.05).结论 ABO血型可能是白血病、淋巴瘤的遗传易感因素,但地理因素可能是主要影响因素之一.     

The Epstein-Barr virus thymidine kinase does not phosphorylate ganciclovir or acyclovir and demonstrates a narrow substrate specificity compared to the herpes simplex virus type 1 thymidine kinase

The Epstein-Barr virus (EBV) thymidine kinase (TK) was expressed in mammalian 143B TK- cells to investigate its substrate specificity. The herpes simplex virus type 1 (HSV-1) TK was similarly expressed for comparison. Both viral TKs conferred a TK+ phenotype on 143B TK- cells. The nucleoside analog ganciclovir (GCV) did not affect the growth of 143B EBV TK or 143B TK- cells but effectively killed 143B HSV-1 TK cells. Furthermore, lysates of 143B EBV TK cells could not phosphorylate GCV, which was confirmed by high-performance liquid chromatography. EBV TK, HSV-1 TK, and EBV TK N-, a truncated EBV TK missing 243 N-terminal amino acids, were purified as fusion proteins expressed in bacteria, and all had TK activity. In addition, EBV TK was observed to have a thymidylate kinase activity but could not phosphorylate GCV, acyclovir, or 2'-deoxycytidine. In competition assays, only nucleoside analogs of thymidine significantly inhibited thymidine phosphorylation by EBV TK, with the following rank order: 5-bromodeoxyuridine > zidovudine > stavudine > sorivudine. These results demonstrate that EBV TK substrate specificity is narrower than those of alphaherpesvirus TKs and that thymidine analogs may be the most suitable nucleoside antivirals to target the enzyme. Clinical implications for gammaherpesviruses are discussed.