Low proportion of G0-phase cells during induction chemotherapy correlates with subsequent remission in acute myeloid leukemia.

Kinetic resistance is assumed to be one of the main mechanisms of drug resistance in acute myeloid leukemia (AML), but the relationship between cell cycle status at diagnosis and achievement of complete remission (CR) is controversial. Based on the possibility that the cell cycle data after starting induction chemotherapy are more important than the pretreatment data, we used 3-color flow cytometry to examine the cell cycle (G0, G1, S, and G2/M phases) of AML cells on days 0, 5, and 9 of the first induction chemotherapy in 20 patients. Cell cycle data at these 3 time points were compared in the patients who achieved CR (CR cases) and in the patients who had persistent leukemia (non-CR cases) after the induction chemotherapy. In the CR cases, there was a tendency for the percentages of G0-phase AML cells on days 5 and 9 to be smaller than that on day 0, while the opposite tendency was observed in the non-CR cases. When cell cycle data were compared between the CR and non-CR cases, the percentage of G0-phase AML cells on day 9 differed significantly (CR cases 6.9% +/- 10.9%, non-CR cases 50.1% +/- 38.4%; P = .0024). This significance remained when the patients' AML subtype was taken into consideration. None of the other cell cycle data at each time point or the hematologic parameters, which may be related to CR achievement, showed differences between the CR and non-CR cases. We emphasize the importance of cell cycle analysis after initiating therapy and suggest that such analysis can identify refractory AML subjects. The identified subjects may be candidates for clinical trials of cell cycle modulators.

Hypokalemic periodic paralysis associated with hypophosphatemia in a patient with hyperinsulinemia.

A 34-year-old man was admitted to the hospital because of acute quadriplegia. On admission, serum potassium was 2.1 mEq/L and serum inorganic phosphate was 1.4 mg/dL. Thyroid function was normal. Serum levels of aldosterone, cortisol, and intact parathyroid hormone were normal. Fasting plasma glucose was 109 mg/dL, and fasting serum insulin was 25.0 U/mL. Shortly after intravenous supplementation of potassium, muscle strength was normalized. Oral glucose tolerance test revealed impaired glucose tolerance and hyperresponse of insulin. During the oral glucose tolerance test, serum potassium and phosphate decreased significantly. These findings suggest that hyperinsulinemia and insulin-induced transmembrane shift of extracellular potassium and phosphate may have been involved in the abnormalities of serum electrolytes and development of hypokalemic periodic paralysis in the present patient.

Repeated cycles of G-CSF-combined postremission chemotherapy for acute myeloid leukemia in a first complete remission: a pilot study.

The cure rate of acute myeloid leukemia might increase if G-CSF were given concurrently with repeated postremission chemotherapy. However, this therapy might cause severe complications, including depletion of normal hematopoietic progenitors as a long-term toxicity. Thus, we conducted a pilot study of this strategy. Twenty-six acute myeloid leukemia patients in a first complete remission (CR) were treated with two courses of consolidation chemotherapy (10-day BHAC-DMP, consisting of behenoyl cytosine arabinoside, daunorubicin, 6-mercaptopurine and prednisolone) and repeated maintenance-intensification therapy including eight cycles of six-day BHAC-DMP. G-CSF (filgrastim) was administered concurrently with these BHAC-DMP therapies. Toxicity during the therapeutic period was not significant in the study group compared with the historical control, treated with the same regimen without G-CSF. Neutrophil recovery after the consolidation therapy was more rapid in the study group than in the historical control (p = 0.066 and 0.024 for the first and second consolidation courses, respectively). Long-term toxicity, such as cytopenia, has not been seen in eight patients who have remained in CR for a long period (range: 39-58 months). At a median follow-up of 39 months, the predicted rate of 42-month CR duration for these 26 patients was 50% (95% confidence limits: 30% to 71%). We conclude that G-CSF-combined repeated BHAC-DMP postremission therapy is feasible. Full elucidation of the clinical benefit of this strategy will require further study.

Pulmonary toxicity after granulocyte colony-stimulating factor-combined chemotherapy for non-Hodgkin's lymphoma.

Sporadic cases have developed pulmonary toxicity after receiving chemotherapy and granulocyte colony-stimulating factor (G-CSF). However, because such cases received chemotherapy that alone frequently causes pulmonary toxicity, the role of G-CSF in this toxicity has been unclear. CHOP therapy (cyclophosphamide, doxorubicin, vincristine and prednisolone) only slightly induces pulmonary toxicity. However, we observed a considerable incidence of this toxicity in non-Hodgkin's lymphoma subjects receiving CHOP therapy and G-CSF (6 out of 52 subjects, 11.5%). In this cohort, among various characteristics, including the dose and interval of CHOP therapy, only the mean peak leucocyte count (MPLC) with each therapy cycle was associated with development of this toxicity (MPLC > or = 23.0 x 10(9) l(-1), 6 out of 29 cases; MPLC < 23.0 x 10(9) l(-1), 0 out of 23 cases; P = 0.020). These findings suggest that the effect of G-CSF is the main determinant of the pulmonary toxicity in these cases. Because the toxicity was associated with a large MPLC and did not recur in cases readministered G-CSF, an idiosyncratic reaction to G-CSF is unlikely to be the pathogenesis of this toxicity. Thus, lowering the G-CSF dose seems to be useful in the prevention of this toxicity. In all six cases, the time course of manifestation of the toxicity was the same, and early application of high-dose corticosteroid led to cure. This knowledge will be helpful in the care of similar cases.

Spontaneous healing of pancreatic abscess after fistulization to the duodenal bulb.

A 70-year-old man was admitted to the hospital because of sudden, upper abdominal and back pain. Laboratory and image data indicated acute pancreatitis. Shortly after the admission, pancreatic and liver abscess with bacteremia developed. Antibiotic therapy seemed effective. A month later, spontaneous fistulization of the pancreatic abscess to the duodenal bulb was found by gastroduodenal fiberscopy. Injection of contrast medium into the duodenal orifice showed that the fistula was draining the abscess and that no other fistula formed from the abscess. Endoscopic retrograde cholangiopancreatogram indicated no fistula formation to the pancreatic duct. The pancreatic abscess became smaller and was not visible using computerized tomography and ultrasonography 3 months later and thereafter. Closure of the duodenal orifice was ascertained by the endoscopy. It is suggested that retrograde infection from the fistula was prevented by the single fistulization to the acidic duodenal bulb, which is not supposed to allow most bacterial growth. Pancreatic abscess usually necessitates operative treatment, even with fistulization to the alimentary tract. It seems likely that the single, small fistulization to the bulb, in addition to the lack of underlying disease and medical and nutritional support, facilitated the spontaneous healing process.

Cell cycle modulation by hematopoietic growth factors in myelodysplastic syndromes: analysis by three-color flow cytometry.

Investigations of the effects of hematopoietic growth factors (HGFs) on the cell cycle of cells from myelodysplastic syndrome (MDS) have been hampered by technical difficulties. In this study, using a recently established flow cytometric method that enables detailed analysis of the cell cycle (Gzero-, G1-, S-, and G2/M-phases) of target cells in a heterogeneous cell population, we examined the effects of granulocyte colony-stimulating factor (G-CSF) and other HGFs on the cell cycle of CD13-positive cells (blasts and other malignant myelocytic and monocytic cells) in MDS. The cell cycle response to G-CSF (decrease in Gzero-phase cells and increase in S-phase cells) was heterogeneous among MDS cases. When the data for 13 MDS cases and 15 de novo AML cases were compared statistically, the magnitude of cell cycle activation by G-CSF was weaker for the cells from the MDS cases. Stem cell factor, interleukin-3, or a combination of these HGFs with G-CSF reduced the Gzero-phase cell percentage in all examined MDS cases whose cell cycle was unresponsive to G-CSF alone. When cytosine arabinoside was added to cells with or without stimulation by HGFs, the viable G0-phase cell count was reduced in HGF-stimulated cells compared with unstimulated cells in seven of eight cases. The present results suggest that G-CSF-induced cell cycle stimulation of malignant cells can be expected in a fraction of MDS patients and that even in MDS patients whose cells do not respond to G-CSF, employment of other HGFs and their combination with G-CSF is worth consideration. The results also suggest that a well-designed therapy using HGFs and chemotherapeutic drugs may reduce the quiescent (Gzero) cell count in MDS, which is assumed to be responsible for drug resistance derived from cell kinetics.

Plasma soluble interleukin-2 receptor level in patients with primary myelodysplastic syndromes: a relationship with disease subtype and clinical outcome.

To assess the hypothesis that the plasma soluble interleukin-2 receptor (sIL-2R) level may have predictive value for morbidity/mortality in patients with myelodysplastic syndromes (MDS), we determined in plasma sIL-2R level of 80 MDS patients and examined their subsequent clinical course. Compared with low-risk MDS (refractory anaemia (RA) and RA with ringed sideroblasts) patients and normal subjects, the plasma sIL-2R level was significantly elevated in high-risk MDS (three other MDS subtypes and acute leukaemia following MDS) patients (high-risk MDS versus low-risk MDS, P < 0.01; high-risk MDS versus normal subjects, P < 0.01). 14/40 low-risk MDS patients developed at least one of the following during the follow-up period: erythrocyte transfusion dependence, infections requiring hospitalization, disease progression or MDS-related death. The plasma sIL-2R level was higher in these eventful subjects than in event-free low-risk subjects (P < 0.0001), and all of 10 low-risk subjects with a plasma sIL-2R level > 540 U/ml experience at least one event. By logistic regression analysis of various parameters in these 40 low-risk subjects, the plasma sIL-2R level was identified as the strongest independent parameter for predicting eventful subjects (P < 0.0047). The plasma sIL-2R level did not show a predictive value in high-risk MDS. This study revealed that the plasma sIL-2R level is significantly elevated in high-risk MDS and suggested that the plasma sIL-2R level is a valuable predictive factors for the clinical outcome in low-risk MDS.

Autoimmune hemolytic anemia in patients with de novo acute myelocytic leukemia.

Autoantibody against erythrocytes has occasionally been observed in patients with de novo acute myelocytic leukemia (AML). However, it is not clear whether this autoantibody in AML patients induces frank hemolysis (autoimmune hemolytic anemia, AIHA), as seen in lymphoid neoplasms. We present two de novo AML patients who showed hemolysis due to antiglobulin test-positive and test-negative AIHA, respectively. AIHA should be considered as one cause of anemia in de novo AML patients, and blood transfusions should be given carefully in such cases to avoid harmful hemolysis.

Renal refractoriness to phosphaturic action of parathyroid hormone in a patient with hypomagnesemia.

A 50-year-old tetanic woman with hypomagnesemia is described. She had partial resection of the stomach and the jejunum at the age of 20 years. Lack of parathyroid hormone (PTH) function was indicated by hypocalcemia, hyperphosphatemia and high tubular reabsorption of phosphate. However, both plasma concentration of PTH and nephrogenous cAMP were normal. Administration of magnesium sulfate completely normalized serum phosphate and tubular transport of phosphate with only a modest increase in nephrogenous cAMP. The present findings suggest that phosphaturic action of PTH is impaired in magnesium deficiency and that steps distal to cAMP production may be responsible for the renal refractoriness to the hormonal action.

Lineage-unrestricted hematologic response to granulocyte colony-stimulating factor in a patient with refractory anemia with excess blasts.

We report a patient with refractory anemia with excess blasts who showed a lineage-unrestricted hematologic response to granulocyte colony-stimulating factor (G-CSF). After 17 months of a stable disease state, the patient developed pneumonia, progression of cytopenia, and reduced cellularity and blast mass in the bone marrow. He was given G-CSF to overcome the pneumonia. Not only the neutrophil count, but also the platelet count increased soon after initiation of the G-CSF therapy; both counts became normal on the fifth day of the G-CSF therapy. Additionally, the anemia improved gradually. The neutrophil and platelet counts were maintained in the normal range for 3 months after cessation of the G-CSF. In vitro studies showed that G-CSF alone stimulated megakaryocyte colony formation from bone marrow mononuclear cells (BMMNC), and accessory cells in the BMMNC were necessary for expression of this G-CSF-induced in vitro megakaryocytopoiesis. These results suggest that, in coordination with accessory cells, G-CSF stimulated megakaryocytopoiesis in the patient. This case provides valuable information for understanding the mechanisms of a lineage-unrestricted hematologic response to G-CSF, which is very rarely observed in MDS.

Defective natural killer (NK) cell-mediated cytotoxicity does not imply clonal involvement of NK cells in myelodysplastic syndromes.

The clonality of purified cells was examined in 10 myelodysplastic syndromes (MDS) patients by analysing the restriction fragment length polymorphism and methylation pattern of the phosphoglycerate-kinase gene. Natural killer (NK) cell-mediated cytotoxicity was also examined. The granulocytes and monocytes were monoclonal or oligoclonal in all cases, except for the monocytes in one case. Conversely, the NK and T cells had a polyclonal pattern in most cases, including all cases who had defective NK cell-mediated cytotoxicity. The hypothesis that reduced NK cell-mediated cytotoxicity in MDS is caused by a clonal involvement of NK cells was not supported by the present study.

Possible gliclazide-induced water retention with azotemia.

An 80-year-old woman with diabetes mellitus was treated with gliclazide. Prior to the gliclazide administration, her urinary excretion of albumin, serum urea nitrogen and serum creatinine were normal. After the medication, oliguria, edema and azotemia developed. On the twenty-fourth day when the edema was severe and generalized, gliclazide administration was terminated. On the following day urinary volume increased suddenly (5,740 ml/day). Polyuria persisted for five days. Edema improved and urea nitrogen and creatinine were normalized thereafter. Though the mechanism is not known, the clinical course suggests that gliclazide is the principal causative factor in the water retention and azotemia in this patient.

Case report: silent thyroiditis developed during alpha-interferon therapy.

Alpha-interferon (IFN-alpha) was used for the treatment of chronic active hepatitis C in a 30-year-old woman who was euthyroid but had low titers of antithyroid antibodies before treatment. Two months after the initiation of IFN-alpha therapy she became thyrotoxic. She had nontender diffuse goiter. A laboratory examination revealed elevated levels of serum free thyroid hormones and a suppressed concentration of serum thyrotropin. Titers of antimicrosomal antibodies increased. The anti-thyrotropin receptor antibody was negative. A 99mTcO- scintigram of the thyroid showed reduced uptake. During the IFN therapy free thyroid-hormone levels started to decline. The IFN-alpha therapy was completed 1 month after the onset of thyrotoxicosis. Two months after the completion of the therapy the patient became euthyroid and 99mTcO- uptake was normalized. It is likely that preexisting chronic thyroiditis was exacerbated to cause silent thyroiditis during IFN-alpha therapy. None of the other 11 patients with chronic hepatitis C who had had no anti-thyroid antibodies and were treated with IFN-alpha showed anti-thyroid antibodies and thyroid dysfunction after the therapy. It is advisable to assess anti-thyroid antibodies and thyroid function in patients who are going to receive IFN-alpha treatment.

A case of rheumatoid arthritis associated with silent thyroiditis.

A 41-year-old female with rheumatoid arthritis had nontender enlarged thyroid gland. Thyroid function tests revealed increased concentrations of serum free T3 (FT3, 10.8 pmol/L) and free T4 (FT4, 31.1 pmol/L) with suppressed concentration of thyrotropin (TSH, lower than 0.1 mU/L) and low 24-hour thyroidal radioactive iodine uptake (1.6%). Serum thyrotropin receptor antibody (TRAb) was negative (0%) and she had positive anti-thyroglobulin and anti-microsomal antibodies. A diagnosis of silent thyroiditis was made based on laboratory findings. Serum concentrations of FT3 and FT4 normalized one month later without treatment. The causal relationship between the two diseases is discussed.

Differential regulation of multiple c-erbA expression by thyrotropin, insulin and insulin-like growth factor I in rat thyroid FRTL-5 cells.

Regulation of multiple c-erbA gene expression was studied in rat thyroid FRTL-5 cells. Two species of erbA alpha (alpha 1 and alpha 2) mRNA and one species of erbA beta (beta 1) mRNA were identified by Northern blot analysis. Withdrawal of thyrotropin (TSH), insulin and serum from the complete medium resulted in an increase in alpha 1 and alpha 2 erbA mRNA levels without altering the level of erbA beta 1 mRNA. Readdition of TSH, N6,2'-O-dibutyryl cAMP or forskolin caused a transient reduction of alpha 1 and alpha 2 mRNA levels (75-90%) at 3-12 h. The alpha 1 and alpha 2 mRNA levels were restored at 24 h. The TSH action was dose-dependent showing the half-maximal effect at around 10(-9) M. Readdition of TSH did not show any effect on beta 1 mRNA level. The action of TSH was not dependent on ongoing protein synthesis but required ongoing transcription. Inhibitors of thyroid hormone biosynthesis, propylthiouracil and methylmercaptoimidazole, did not show any effect on TSH action. Readdition of insulin or insulin-like growth factor I (IGF-I) caused a dose-dependent reduction of alpha 1 and alpha 2 mRNA levels without any effect on beta 1 mRNA level. Their action was slower than TSH and persistent. The actions of insulin and IGF-I were dependent on both ongoing translation and transcription. These results indicate that TSH and insulin/IGF-I reduce levels of c-erbA alpha 1 and alpha 2 mRNA possibly by two distinct mechanisms without altering c-erbA beta 1 mRNA level in FRTL-5 cells.

Thyroid hormone metabolism and nuclear binding in Gunn rats.

We examined the serum concentrations of total, free thyroid hormones and TSH, activity of hepatic T4 5'-deiodinase, and T3 binding to hepatic nuclei in homozygous (j/j) and heterozygous (j/+) Gunn rats. Both total T3 and free T3 (FT3) concentrations in sera from j/j rats were significantly lower than those of j/+ rats on 5-10, 15-20, and 25-30 days after birth. Both total T4 and free T4 (FT4) concentrations in j/j and j/+ rat sera were not significantly different on 5-10 days. However, in j/j rats they were significantly higher than those of j/+ rats on days 15-20 and 25-30. Serum reverse T3 (rT3) concentrations were higher in j/j than in j/+ rats on days 5-10, 15-20, and 25-30. Serum TSH concentration in j/j and j/+ rats on 15 days post-natal were 1.42 +/- 1.28 and 1.65 +/- 1.24 micrograms/l (mean +/- SD), respectively, which were not significantly different from each other. T3 formation from T4 in hepatic microsomal fractions obtained 15 days after birth was significantly lower in homozygotes than in heterozygotes (4.89 +/- 1.18 vs 11.15 +/- 2.38 pmol/mg protein/min, p less than 0.005). Binding constants (Ka) as well as maximal binding capacities (MBC) for T3 of hepatic nuclei from 15 day-old j/j and j/+ rats were similar (ka; 3.58 x 10(9) vs 3.15 x 10(9) M-1, MBC; 0.316 vs 0.380 pmol/mg DNA). From these results we suggest that decreased conversion from T4 to T3 is one of the major reasons for high serum levels of T4 and rT3, and low levels of T3 in j/j rats, and that nuclear T3 binding and pituitary TSH secretion are unaltered in j/j rats.

The deoxyribonucleic acid regions involved in the hormonal regulation of thyroglobulin gene expression.

Transcription of the thyroglobulin (TG) gene is stimulated by TSH via cAMP. We have characterized the sequence elements responsible for the hormone-dependent expression of TG gene in rat thyroid FRTL-5 cells using internal deletion and linker-scanning mutants of the minimal TG promoter (-170 basepairs) fused with the bacterial chloramphenicol acetyltransferase reporter gene. The TG gene is regulated by at least two regions located between -165 and -140 bp (TG-III) and between -95 and -65 bp (TG-I) from the transcription initiation site. The intervening region can be deleted without significant effect on the promoter activity. Either of the two regions alone does not promote hormone-dependent transcription. A DNase footprinting assay showed that TG-I and TG-III are the principal protein-binding sites and that the proteins interacting with these two regions are induced by TSH or cAMP. These results suggest that the hormone-dependent expression of TG gene may be achieved by cooperative interaction of the proteins bound to TG-I and TG-III.

Similar nuclear factors mediate stimulation of rat thyroglobulin gene transcription by thyrotropin and insulin-like growth factor-I.

Transcription of the thyroglobulin (TG) gene in rat thyroid FRTL-5 cells is stimulated by two hormones, TSH and insulin-like growth factor-I (IGF-I). The effect of TSH is mimicked by cAMP. Promoter regions of the rat TG gene responsible for hormonal action as well as the nuclear regulatory proteins that interact with these regions were characterized. Minimal promoter that responds to both hormones has been found to be up to -171 basepairs from the transcription initiation site. In DNase-I footprinting analysis, nuclear extracts from cells treated with either of these hormones protected the same two major regions within the minimal promoter. Mutations in these two regions abolished basal, TSH-stimulated, as well as IGF-I-stimulated expression of the fused reporter gene chloramphenicol acetyltransferase. DNA mobility shift assay revealed that cAMP and IGF-I induce binding of similar nuclear proteins to these promoter regions. These results suggest that rat TG gene transcription is regulated by the convergent action of two distinct signaling pathways, possibly involving similar DNA-binding nuclear proteins and regulatory sequences of the TG gene promoter.

Thyroxine binding to human serum albumin immobilized on sepharose and effects of nonprotein albumin-binding plasma constituents.

125I-thyroxine (125I-T4) binding to human serum albumin (HSA) covalently attached onto CNBr-activated Sepharose (HSA-Sepharose) was studied. 125I-T4 binding to HSA-Sepharose was rapid and saturable. Nonlinear curve-fitting analysis of binding isotherms revealed two classes of binding sites. The values of dissociation constants of high and low affinity sites were 2.19 +/- 0.53 x 10(-6) M and 2.69 +/- 0.78 x 10(-5) M, respectively. The number of binding sites of the high and the low affinity sites were 1.28 +/- 0.46 mol/mol and 23.5 +/- 9.7 mol/mol of HSA, respectively. Fatty acids and bilirubin competitively inhibited the high-affinity binding of 125I-T4 to HSA-Sepharose without affecting the low-affinity binding. 8-anilino-1-naphthalene sulfonic acid (ANS) inhibited the high affinity T4 binding via reduction of the binding capacity. Unlabeled T4 showed little inhibition of ANS binding to HSA, as measured by fluorescence intensity. These results suggest that ANS allosterically inhibits the high-affinity T4 binding to HSA-Sepharose.