Manufacture of a weakly denatured collagen fiber scaffold with excellent biocompatibility and space maintenance ability.

Although collagen scaffolds have been used for regenerative medicine, they have insufficient mechanical strength. We made a weakly denatured collagen fiber scaffold from a collagen fiber suspension (physiological pH 7.4) through a process of freeze drying and denaturation with heat under low pressure (1 × 10(-1) Pa). Heat treatment formed cross-links between the collagen fibers, providing the scaffold with sufficient mechanical strength to maintain the space for tissue regeneration in vivo. The scaffold was embedded under the back skin of a rat, and biocompatibility and space maintenance ability were examined after 2 weeks. These were evaluated by using the ratio of foreign body giant cells and thickness of the residual scaffold. A weakly denatured collagen fiber scaffold with moderate biocompatibility and space maintenance ability was made by freezing at -10 °C, followed by denaturation at 140 °C for 6 h. In addition, the direction of the collagen fibers in the scaffold was adjusted by cooling the suspension only from the bottom of the container. This process increased the ratio of cells that infiltrated into the scaffold. A weakly denatured collagen fiber scaffold thus made can be used for tissue regeneration or delivery of cells or proteins to a target site.

Isolated spinal neurosarcoidosis: an autopsy case.

STUDY DESIGN: Autopsy cases of isolated spinal neurosarcoidosis are extremely rare or none. OBJECTIVES: To report an autopsy case of isolated spinal neurosarcoidosis without the involvement of other organs. SUMMARY OF BACKGROUND DATA: A few reports of isolated spinal neurosarcoidosis are present, but no autopsy cases of isolated spinal neurosarcoidosis are present in the English literature. SETTING: Japan. METHODS: An autopsy case of isolated spinal neurosarcoidosis was examined pathologically. RESULTS: A 54-year-old woman was admitted to our hospital because of leg numbness and muscle weakness. Imaging modalities showed an irregular contour and a mass-like lesion in the spinal cord. No biopsies were performed. Clinical diagnoses were suspected sarcoidosis and malignant lymphoma. The patient was treated by steroids and anti-cancer drugs, but she suddenly died of unknown cause. An autopsy revealed fibrosis and non-caseating granulomata in the spinal cord. No acid-fast bacteria or fungi were recognized by special stains. A PCR revealed no acid-fast bacteria including tuberculosis. Other organs including the brain and lung showed no sarcoidosis lesions. A pathological diagnosis of isolated spinal neurosarcoidosis was made. Other pathological diagnoses were systemic congestion, lung emphysema, food impaction in the upper esophagus and larynx, cardiac hypertrophy and marked dilation of the colon. The cause of death was thought to be respiratory failure. CONCLUSION: We have reported a very rare autopsy case of isolated spinal neurosarcoidosis, with an emphasis on pathological findings.

CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells.

We studied the effect of CMC-544, the calicheamicin-conjugated anti-CD22 monoclonal antibody, used alone and in combination with rituximab, analyzing the quantitative alteration of target molecules, that is, CD20, CD22, CD55 and CD59, in Daudi and Raji cells as well as in cells obtained from patients with B-cell malignancies (BCM). Antibody inducing direct antiproliferative and apoptotic effect, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were tested separately. In Daudi and Raji cells, the CDC effect of rituximab significantly increased within 12 h following incubation with CMC-544. The levels of CD22 and CD55 were significantly reduced (P<0.001 in both cells) after incubation with CMC-544, but CD20 level remained constant or increased for 12 h. Similar results were obtained in cells from 12 patients with BCM. The antiproliferative and apoptotic effect of CMC-544 were greater than that of rituximab. The ADCC of rituximab was not enhanced by CMC-544. Thus, the combination of CMC-544 and rituximab increased the in vitro cytotoxic effect in BCM cells, and sequential administration for 12 h proceeded by CMC-544 was more effective. The reduction of CD55 and the preservation of CD20 after incubation with CMC-544 support the rationale for the combined use of CMC-544 and rituximab.

Efficacy of gemtuzumab ozogamicin on ATRA- and arsenic-resistant acute promyelocytic leukemia (APL) cells.

Acute promyelocytic leukemia (APL) cells express a considerable level of CD33, which is a target of gemtuzumab ozogamicin (GO), and a significantly lower level of P-glycoprotein (P-gp). In this study, we examined whether GO was effective on all-trans retinoic acid (ATRA)- or arsenic trioxide (ATO)-resistant APL cells. Cells used were an APL cell line in which P-gp was undetectable (NB4), ATRA-resistant NB4 (NB4/RA), NB4 and NB4/RA that had been transfected with MDR-1 cDNA (NB4/MDR and NB4/RA/MDR, respectively), ATO-resistant NB4 (NB4/As) and blast cells from eight patients with clinically ATRA-resistant APL including two patients with ATRA- and ATO-resistant APL. The efficacy of GO was analyzed by (3)H-thymidine incorporation, the dye exclusion test and cell cycle distribution. GO suppressed the growth of NB4, NB4/RA and NB4/As cells in a dose-dependent manner. GO increased the percentage of hypodiploid cells significantly in NB4, NB4/RA and NB4/As cells, and by a limited degree in NB4/MDR and NB4/RA/MDR cells. Similar results were obtained using blast cells from the patients with APL. GO is effective against ATRA- or ATO-resistant APL cells that do not express P-gp, and the mechanism of resistance to GO is not related to the mechanism of resistance to ATRA or ATO in APL cells. Leukemia (2005) 19, 1306-1311. doi:10.1038/sj.leu.2403807; published online 26 May 2005.

Transrectal colour Doppler ultrasonography for quantifying angiogenesis in prostate cancer.

OBJECTIVE: To assess the correlation between angiogenesis and Doppler signal intensity using transrectal colour Doppler ultrasonography (CDUS) in patients with prostate cancer. PATIENTS AND METHODS: The study comprised 56 patients who underwent radical prostatectomy and had untreated tumours with a volume of> 0.1 mL in the peripheral zone. CDUS images were recorded on videotape before surgery. The Doppler signal intensity in tumours was evaluated using the colour pixel intensity (PI). Microvessel density (MVD) and vascular endothelial growth factor (VEGF) immunoreactivity were determined in the prostatectomy specimens. Microvessels were identified by immunohistochemical staining of endothelial cells for CD31. RESULTS: The PI in the tumour correlated with MVD (P < 0.001) and increased with higher levels of VEGF immunoreactivity (P = 0.004). There was no correlation between Gleason score and MVD or PI in the tumour. CONCLUSION: Blood flow assessed by CDUS may reflect the state of angiogenesis in prostate cancer. CDUS may be a useful technique for predicting tumour progression or prognosis, and may be useful for monitoring the effects of anti-angiogenic agents in the future.

Possible dominant-negative mutation of the SHIP gene in acute myeloid leukemia.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) is crucial in hematopoietic development. To evaluate the possible tumor suppressor role of the SHIP gene in myeloid leukemogenesis, we examined primary leukemia cells from 30 acute myeloid leukemia (AML) patients, together with eight myeloid leukemia cell lines. A somatic mutation at codon 684, replacing Val with Glu, was detected in one patient, lying within the signature motif 2, which is the phosphatase active site. The results of an in vitro inositol 5'-phosphatase assay revealed that the mutation reduced catalytic activity of SHIP. Leukemia cells with the mutation showed enhanced Akt phosphorylation following IL-3 stimulation. K562 cells transfected with the mutated SHIP-V684E cDNA showed a growth advantage even at lower serum concentrations and resistance to apoptosis induced by serum deprivation and exposure to etoposide. These results suggest a possible role of the mutated SHIP gene in the development of acute leukemia and chemotherapy resistance through the deregulation of the phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3)/Akt signaling pathway. This is the first report of a mutation in the SHIP gene in any given human cancer, and indicates the need for more attention to be paid to this gene with respect to cancer pathogenesis.

Regenerative repair of the mandible using a collagen sponge containing TGF-beta1.

INTRODUCTION: Alveolar bone resorption and atrophy of the mandible are a major challenge for regeneration medicine. In the present investigation, a collagen sponge that contained TGF-beta1 was placed at a mandibular defect and the osteogenic effects of collagen-TGF-beta1, complex were evaluated. MATERIAL AND METHODS: The Pm2, Pm3, and Pm4 teeth on both sides of the mandibles of 12 adult beagle dogs (9.0-12.0 kg) were extracted. After the extraction-site wounds healed, a bone defect (10.0 x 15.0 mm-wide, 10.0 mm-deep or 10.0 x 10.0 mm-wide, 10.0 mm-deep) was created on the mandible. A collagen sponge (10.0 x 10.0 x 10.0 mm) that contained TGF-beta1 (1.0 microg, 5.0 microg, or 10.0 microg, in physiological saline) was placed at the bottom of the defect and the overlying mucous membrane was sutured with 4-0 prolene. As a control, a collagen sponge that contained physiological saline only was placed in a defect on the opposite side. Two weeks after the surgery the wounds above the bone defects on both the control and TGF-beta1-treated sides had healed completely. RESULTS: At four, six, or eight weeks post-operatively animals were killed. Soft X-ray and bone-salt measurement analyses confirmed clearly that there was greater calcified bone formation in the defects into which TGF-beta1 had been incorporated than with the control defects. The implanted collagen sponges were fully resorbed and the bone tissue had regenerated from the bottom of the defects on the TGF-beta1, side by four weeks. On the control side, no such regeneration was observed. CONCLUSIONS: These results indicate that TGF-beta1, released slowly from a collagen sponge was effective in promoting bone remodeling when applied to mandibular defects in adult dogs.

Modulation of heat-induced cell death in PC-3 prostate cancer cells by the antioxidant inhibitor diethyldithiocarbamate.

OBJECTIVE: To examine the relationships between the form of cell death (apoptosis or necrosis), reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and the level of heat-shock protein 70 (hsp 70) expression after thermotherapy of PC-3 prostate cancer cells; also assessed were the tumoricidal effects of combined treatment with both heat and the antioxidant inhibitor diethyldithiocarbamate (DDC). MATERIALS AND METHODS: PC-3 cells were treated with thermotherapy at 42, 43 or 44 degrees C for 30, 60, 90 or 120 min. Cell proliferation, ROS generation, SOD activity and cellular hsp 70 level were determined using tetrazolium-based cytotoxicity, fluorescent dichlorofluorescein (DCF) and nitroblue tetrazolium assays, Western blot analysis and flow cytometry, respectively. The apoptotic and necrotic cells were determined by staining with propidium iodide and fluorescein isothiocyanate-labelled annexin V. These variable were also measured after combined treatment of PC-3 cells with 1 mmol/L DDC and thermotherapy at 43 or 44 degrees C for 60 min. RESULTS: Cell survival was significantly lower after heating cells at 43 degrees C for 60, 90 and 120 min and at 44 degrees C for all periods tested (P<0.05). At 43 degrees C apoptosis increased with the duration of heating and was similarly enhanced after heating at 44 degrees C for 30 min. Necrosis was not increased by heating at 42 or 43 degrees C, but was markedly enhanced after heating at 44 degrees C with both the duration of heating and with time after heating. Significant increases in DCF production were induced by heating at 43 degrees C for 60, 90 and 120 min (P<0.05) and at 44 degrees C at all times (P<0.010-0.005). There was a significant correlation between the level of ROS generation and necrosis (P<0.001) but no correlation between the ROS level and apoptosis. SOD activity increased in cells after heating at 43 degrees C, with significant differences among cells heated for 60, 90 and 120 min (P<0.05). After heating at 44 degrees C, SOD activity was maximal in cells heated for 30 min (P<0.005), by 30 min and then decreased with time after heating. There were significant increases in hsp 70 level in cells heated at 43 degrees C for 90 and 120 min (P<0.05) and at 44 degrees C for 30 and 60 min (P<0.05 and <0.025, respectively). Hsp 70 levels decreased after heating at 44 degrees C for 90 and 120 min. The combination of DDC and heating significantly increased ROS generation and the percentage of cell death, and decreased SOD activity (P<0.05). CONCLUSION: These findings show a qualitative change in the form of cell death induced by thermotherapy of PC-3 cells, which changed from apoptosis to necrosis according to the degree and duration of heating. Mild thermotherapy induced marginally low occurrence of apoptosis of PC-3 cells and DDC may represent a useful future strategy for the treatment of prostate carcinoma.

Reduced effect of gemtuzumab ozogamicin (CMA-676) on P-glycoprotein and/or CD34-positive leukemia cells and its restoration by multidrug resistance modifiers.

Gemtuzumab ozogamicin (CMA-676), a calicheamicin-conjugated humanized anti-CD33 mouse monoclonal antibody, has recently been introduced clinically as a promising drug for the treatment of patients with acute myeloid leukemia (AML), more than 90% of which express CD33 antigen. However, our recent study suggested that CMA-676 was excreted by a multi- drug-resistance (MDR) mechanism in P-glycoprotein (P-gp)-expressing leukemia cell lines. We analyzed the in vitro effects of CMA-676 on leukemia cells from 27 AML patients in relation to the amount of P-gp, MDR-associated protein 1 (MRP1), CD33 and CD34, using a multi-laser-equipped flow cytometer. The cytocidal effect of CMA-676, estimated by the amount of hypodiploid portion on cell cycle, was inversely related to the amount of P-gp estimated by MRK16 monoclonal antibody (P = 0.004), and to the P-gp function assessed by intracellular rhodamine-123 accumulation in the presence of PSC833 or MS209 as a MDR modifier (P = 0.0004 and P = 0.002, respectively). In addition, these MDR modifiers reversed CMA-676 resistance in P-gp-expressing CD33(+) leukemia cells (P = 0.001 with PSC833 and P = 0.0007 with MS209). In CD33(+) AML cells from 13 patients, CMA-676 was less effective on CD33(+)CD34(+) than CD33(+)CD34(-) cells (P = 0.002). PSC833 partially restored the effect of CMA-676 in CD33(+)CD34(+) cells. These results suggest that the combined use of CMA-676 and a MDR modifier will be more effective on CD33(+) AML with P-gp-related MDR.

Arsenic trioxide therapy for relapsed or refractory Japanese patients with acute promyelocytic leukemia: need for careful electrocardiogram monitoring.

Recent studies have shown that arsenic trioxide (As(2)O(3)) can induce complete remission in patients with acute promyelocytic leukemia (APL). We tested the efficacy and safety of As(2)O(3) for the treatment of patients with APL who had relapsed from or become refractory to all-trans retinoic acid (ATRA) and conventional chemotherapy in a prospective study. As(2)O(3) at a dose of 0.15 mg/kg was administered until the date of bone marrow remission to a maximum of 60 days. In patients who achieved complete remission (CR), one additional course of As(2)O(3) was administered using the same dose for 25 days. Of 14 patients, 11 (78%) achieved CR. Six of 10 patients who achieved CR showed disappearance of PML-RARalpha transcript by RT-PCR assay. The duration of As(2)O(3)-induced CR ranged from 4 to 22 months (median, 8 months) at a median follow-up of 17 months. Adverse events included 13 electrocardiogram abnormalities (13 QTc prolongation, eight ventricular premature contraction, four nonsustained ventricular tachycardia and two paroxysmal supraventricular tachycardia), seven nausea and vomiting, four pruritus, three peripheral neuropathy, three fluid retention and one APL differentiation syndrome. Four patients received antiarrhythmic agents. Hyperleukocytosis developed in five patients and in three cytotoxic drugs were necessary. Other adverse events were relatively mild. As(2)O(3) treatment is effective and relatively safe in relapsed or refectory patients with APL. Cardiac toxicities in patients with QTc prolongation should be carefully monitored.

All-trans retinoic acid (ATRA) differentiates acute promyelocytic leukemia cells independently of P-glycoprotein (P-gp) related multidrug resistance.

Here the relationship between all-trans retinoic acid (ATRA)-resistance and P-glycoprotein (P-gp)-associated multidrug resistance (MDR) is discussed in acute promyelocytic leukemia (APL). First, the remission rates of ATRA therapy are similar in relapsed/refractory APL to the preceding chemotherapy given and in newly diagnosed APL. Second, MDR1 cDNA-transduced NB4 (NB4/MDR) cells accumulate less Rhodamine-123 (Rh123) than NB4 cells, but there is no difference in the intracellular ATRA concentration between them. PSC833 or MS209. MDR modifiers, increases the intracellular accumulation of Rh123 in NB4/MDR and APL cells expressing P-gp, but not of ATRA. Third, the expression of CD11b, the NBT reduction activity, the proportion of apoptotic cells and the morphology are not different between NB4/MDR and NB4 cells, and between APL cells expressing P-gp and not. APL cells express little P-gp, and mainly express CD33 but no CD34. Despite previous reports that ATRA-resistant APL cells express more P-gp than ATRA-sensitive ones, P-gp and ATRA-resistance seems to exist independently.

Alterations of beta- and gamma-catenin in N-butyl-N-(-4-hydroxybutyl)nitrosamine-induced murine bladder cancer.

Abnormal degradation of beta-catenin caused by alteration of the glycogen synthase kinase-3beta (GSK-3beta) consensus motif is an important step for carcinogenesis. We hypothesize that beta- and gamma-catenin may play an important role in the pathogenesis of bladder cancer. We tested this hypothesis through analysis of beta- and gamma-catenin in both murine and human bladder cancers. A murine bladder cancer model was prepared by use of N-butyl-N-(-4-hydroxybutyl)nitrosamine (BBN) in 6-week-old male B6D2F1 mice. After 4, 8, 12, 16, 20, 24, and 28 weeks of BBN treatment, bladder specimens were harvested and analyzed for both protein and gene expression for beta- and gamma-catenin. Mutational analysis of the NH(2)-terminal regulatory domains of beta- and gamma-catenin was performed in each specimen by PCR-single-strand conformational polymorphism (SSCP) analysis. Mutations were further confirmed by direct DNA sequencing with a dye terminator method. Human bladder cancer specimens with normal tissues, dysplasia, carcinoma in situ, and carcinoma of grades, 1, 2, and 3 were also analyzed for beta- and gamma-catenin expression. beta- and gamma-catenin were analyzed for mutations by SSCP and direct DNA sequencing. Intracellular accumulation of beta- and gamma-catenin was observed in 6 of 20 invasive carcinoma specimens. There was no intracellular accumulation of beta- and gamma-catenin in mucosal dysplasia, papillary or nodular dysplasia, and carcinoma in situ specimens. On an SSCP analysis for beta-catenin, abnormal bandshifts were detected in two invasive carcinomas with intracellular beta-catenin accumulation. Further sequencing revealed two mutations [AGT(S) to ATT(I) and TCT(S) to CCT(P)] within the consensus motif for GSK-3beta phosphorylation. On the other hand, SSCP analysis for gamma-catenin followed by sequencing revealed three mutations in two invasive carcinomas with intracellular accumulation of gamma-catenin. These three alterations affected the 3' downstream region outside the GSK-3beta phosphorylation site [ACC(T) to GCC(A), CTC(L) to ATC(I), and CTC(L) to ATG(M)]. In human bladder cancer, beta- and gamma-catenin expression was significantly weaker than in normal bladder. On SSCP analysis one abnormal bandshift was observed in high-grade human bladder cancer with intracellular beta-catenin accumulation. DNA sequencing revealed mutation TCT(S) to TGT(C). In summary, alterations in beta- and gamma-catenin are late events favoring tumor progression in mouse BBN-induced bladder cancer. Changes affecting the GSK-3beta phosphorylation site appear to be associated with activation of beta-catenin, but not with activation of gamma-catenin. In human blabber cancer, beta- and gamma-catenin expression is similar to the expression in the mouse model. The present study demonstrates that beta- and gamma-catenin may play an important role in bladder cancer progression.

Multilocular spermatocele: a case report.

We describe a case of a multilocular spermatocele. Ultrasound examination revealed several cystic spaces at the head of the left epididymis. Epididymal tumor could not be excluded, and therefore surgical exploration was performed. Histopathological examination of the specimen revealed a multilocular spermatocele arising from the rete testis. Most spermatoceles remain small and rarely present marked clinical problems. but they are occasionally large, and may simulate a solid tumor.

Factor analysis of the components of 12 standard test batteries, for unilateral spatial neglect, reveals that they contain a number of discrete and important clinical variables.

The aim of this study was to investigate a conventional battery of tests capable of assessing the presence of the component and extent of the lesions in patients with unilateral spatial neglect. Ninety-four patients who had unilateral spatial neglect with a stroke in right hemisphere were assessed on 12 traditional neglect batteries 4 weeks after the onset. Computerized tomography was also performed to investigate the possible anatomical relationships with each neglect battery. Factor analysis showed that the tests loaded significantly on five factors. There are not only visual scanning factors but also factors of imaging, visual judgement, visual cognition and effectiveness from left hemisphere in the unilateral spatial neglect. There are high correlations between each neuropsychological test and neglect batteries. Furthermore, lesions in the paraventricular white matter were associated with clock and person drawing tasks. Lesions in the occipital lobe were associated with reading, explaining and visual counting tasks. Lesions in the temporal lobe and the posterior limb of the internal capsule were associated with line bisection tasks. It is suggested that it is possible that there are some different components in unilateral spatial neglect. Failure in some tasks may predict different lesions in terms which include localization.

Prolongation of the QT interval and ventricular tachycardia in patients treated with arsenic trioxide for acute promyelocytic leukemia.

BACKGROUND: Recently, arsenic trioxide has increasingly been used for relapsed acute promyelocytic leukemia. However, it is known to have several adverse effects, including acute cardiac toxicities. OBJECTIVE: To determine cardiac toxicities resulting from arsenic trioxide therapy in patients with relapsed or refractory acute promyelocytic leukemia. DESIGN: Phase II clinical prospective cohort study. SETTING: A university hospital in Hamamatsu, Japan. PATIENTS: 8 patients with relapsed acute promyelocytic leukemia. INTERVENTION: Arsenic trioxide, 0.15 mg/kg of body weight, administered daily by 2-hour infusion for a maximum of 60 days. MEASUREMENTS: Continuous monitoring with ambulatory electrocardiography. RESULTS: Five patients (63%) achieved complete remission. During induction therapy with arsenic trioxide, prolonged QT intervals were observed in all patients. Ventricular premature contractions were noticed during 8 of 12 courses of therapy. Four patients developed nonsustained ventricular tachycardia and required treatment with antiarrhythmic agents. CONCLUSIONS: Cardiac toxicity occurs during arsenic trioxide therapy in patients with acute promyelocytic leukemia. Such patients should be monitored for prolonged QT intervals and ventricular arrhythmia.

Artificial trachea and long term follow-up in carinal reconstruction in dogs.

We have already reported successful carinal reconstruction of the trachea with an observation period of 1 - 2 years. In this study, we evaluate the long-term safety and efficacy of the reconstruction after 5-years of follow-up. The Y-shaped Marlex mesh tube was reinforced with a polypropylene spiral and coated with atelocollagen made from porcine skin. The prosthesis was 60 mm long with an outer diameter of 18 mm. Replacement of the tracheobronchial bifurcation was preformed through a right thoracotomy in a beagle dog. Bronchoscopical examination and sampling of the tracheal epithelium was performed periodically to check the function of cilia. The implanted prothesis was promptly infiltrated by the surrounding connective tissue and completely incorporated by the host trachea and bronchus. Bronchoscopically, sufficient epithelization was confirmed from the upper to the lower site of anastomosis. After 5 years neither stenosis nor dehiscence was observed. In spite of there being mesh-exposure at the luminal surface, the dog had no clinical symptoms until sacrifice for pathological examination. The bent frequency of the cilia was maintained within the normal range, indicating functional recovery of the regenerating airway. Our tracheal prosthesis is promising for clinical repair of the tracheobronchial bifurcation.

Concomitant presence of bladder cancer and neurogenic bladder in a patient with HTLV-1 carrier: a case report.

We describe a case of an HTLV-1 carrier who developed bladder cancer and neurogenic bladder. HTLV-1 is thought to alter host immune function and to contribute to the development of other malignancies. It is also sometimes reported that urinary symptoms precede pyramidal symptoms in patients with HAM. To our knowledge, concomitant presence of bladder cancer and neurogenic bladder in an HTLV-1 carrier has not been previously reported.

In vitro IL-12 treatment of peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes: increase in cytotoxicity and reduction in WT1 gene expression.

Interleukin-12 (IL-12) has potent antitumor activities. We examined whether IL-12 enhanced the cytotoxicity of peripheral blood mononuclear cells (PBMNC) and decreased leukemia cells in 30 patients with leukemia or myelodysplastic syndromes (MDS): 12 acute myeloid leukemia (AML) (five in complete remission (CR) and seven in non-CR); six chronic myeloid leukemia (CML); and 12 MDS (three refractory anemia (RA), eight RA with excess of blasts and one chronic myelomonocytic leukemia). PBMNC from patients and five healthy volunteers were cultured at 5 x 10(5)/ml parallel with or without 100 units/ml of IL-12 for 3 days. Cytotoxicity of PBMNC against K562 cells was assessed by flow cytometry. To quantify the amount of leukemia cells, WT1 mRNA was measured by competitive reverse transcription polymerase chain reaction (RT-PCR), since WT1 mRNA is considered as a marker of minimal residual disease (MRD) in leukemia or MDS. The cytotoxicity of non-IL-12-treated PBMNC of 30 patients was 13.4+/-9.3% at the effector to target (E:T) ratio of 20:1, and significantly lower than that of normal subjects (25.7+/-8.4%). The cytotoxicity increased to 30.6+/-17.9% in the IL-12-treated PBMNC. WT1 mRNA in PBMNC of five healthy volunteers was less than 10(3) copies/microg of total RNA. Following the 3-day IL-12 treatment, mean WT1 mRNA of PBMNC was reduced from 10(4.8) to 10(4.2) copies/microg of total RNA in six CML patients, from 10(5.4) to 10(4.8) copies/microg in 12 MDS patients and from 10(5.0) to 10(4.2) copies/microg in five AML patients in CR, but not reduced in five of seven AML in non-CR. These results showed that IL-12 significantly enhanced PBMNC cytotoxicity and decreased the quantity of leukemia cells in PBMNC of most patients with MDS, CML and AML in CR. IL-12 might be of considerable benefit in the elimination of MRD in patients with hematological malignancies.