Impaired stem cell function of CD34+ cells selected by two different immunomagnetic beads systems.

We have been investigating the hematopoietic stem cell (HSC) activity of peripheral blood-derived CD34(+) cells selected by two different laboratory immunomagnetic beads systems (MiniMACS and Isolex 50). In this study, the quality of purified CD34(+) cells was directly compared using clonal cell culture, a cobblestone area-forming cell (CAFC) assay, and an in vivo severe combined immunodeficiency (SCID)-repopulating cell (SRC) assay. It was found that CD34(+) cells selected by these two immunomagnetic methods showed a reduced yield of colony-forming cells and CAFCs compared with cells enriched by the StemSep device (a negative selection method). However, these CD34(+) cells still showed significant SRC activity, including multilineage lymphomyeloid reconstitution. The percentage of human CD45(+) cells in murine bone marrow after transplanting 5 x 10(5) CD34(+) cells selected by the Isolex 50 was significantly lower than after transplanting cells selected by the MiniMACS or the StemSep. Our findings clearly demonstrated that CD34(+) cells selected by the MiniMACS system had superior HSC functions, including SRC activity, compared with cells separated by the Isolex 50 system. More detailed functional analysis of immunomagnetically separated CD34(+) cells may provide useful knowledge for basic research on HSCs as well as for clinical HSC transplantation.

Human cord blood long-term engrafting cells are CD34+ CD38-.

There have been controversies about CD34 and CD38 expression by human cord blood (CB) stem cells. Using the newborn NOD/SCID/beta2-microglobulin-null mouse assay that we recently developed, we examined the in vivo engrafting capability of human CB cells. Almost all of the 4-5 months engrafting cells were found in CD34(+) population. The capability of secondary reconstitution was found only in the CD34(+) cells. When the CD34(+) CB cells were separated into CD38(-) and CD38(+) subpopulations and tested for engraftment, the majority of the engrafting cells were detected in the CD38(-) subpopulation. These findings are consistent with the results from studies of murine stem cells and strongly indicate that the phenotype of human CB stem cells is CD34(+) CD38(-).

Interleukin-11 (IL-11) enhances clonal proliferation of acute myelogenous leukemia cells with strong expression of the IL-11 receptor alpha chain and signal transducing gp130.

We examined the effect of recombinant human interleukin (IL)-11 alone or in combination with various colony-stimulating factors (CSFs), including IL-3, granulocyte/macrophage (GM)-CSF, granulocyte (G)-CSF, stem cell factor (SCF), flt3 ligand (FL), and thrombopoietin (TPO), on colony formation by leukemic progenitor cells (L-CFU) obtained from 33 patients with acute myelogenous leukemia (AML). Leukemic colony formation was found in approximately 70 to 80% of the patients in the presence of at least one of the above CSFs. Although IL-11 alone did not support L-CFU, the growth of these progenitors in the presence of other cytokines was enhanced by IL-11 in 16 out of 33 patients and it showed a synergistic action with G-CSF in 12 of them. This synergistic action occurred in seven out of nine M5 patients (French-American-British (FAB) classification). A single cell clone-sorting experiment clearly demonstrated that this synergistic effect was operative at the single progenitor cell level. The number of leukemic cells proliferating in the presence of G-CSF+IL-11 was significantly higher than in the presence of G-CSF alone, suggesting that IL-11 recruited dormant leukemic progenitors into the cell cycle. Flow cytometric analysis revealed that all types of AML blast cells (M0 approximately M6) ubiquitously expressed gp130, although the level of expression was significantly higher in M5 cells. In contrast, expression of the IL-11 receptor alpha chain (IL-11Ralpha) varied between FAB types. Blast cells obtained from M1, M3 and M5 patients showed higher levels of expression, with M5 cells showing the strongest expression. Interestingly, the leukemic progenitor cells for which proliferation was synergistically enhanced by IL-11 had significantly higher expression of both IL-11Ralpha and gp130. These results suggest that administration of IL-11 in vivo may stimulate the proliferation of leukemic progenitor cells, particularly M5 cells, in the presence of G-CSF, and that the responsiveness of L-CFU to IL-11 may be predicted by a simple receptor assay.

Human cord blood-derived primitive progenitors are enriched in CD34+c-kit- cells: correlation between long-term culture-initiating cells and telomerase expression.

We studied the functional characteristics of subpopulations of cord blood-derived CD34+ cells expressing different levels of CD38 and c-kit antigens, using clonal cell culture and long-term culture with allogeneic bone marrow stromal cells or the MS-5 murine stromal cell line to assay long-term culture-initiating cells (LTC-IC) in each subpopulation. To investigate the capacity for replication, proliferation, and differentiation of each subpopulation of CD34+ cells, we also studied the correlation between LTC-IC and telomerase activity. After 5 weeks of coculture, LTC-IC accounted for one out of 32 CD34+CD38- cells and one out of 33 CD34+c-kit- cells. In contrast, the frequency of LTC-IC was low in their antigen-positive counterparts (one per 84 CD34+CD38+ cells, one per 90 CD34+c-kit(low) cells, and very low among CD34+c-kit(high) cells). It was noteworthy that some LTC-IC derived from CD34+CD38- as well as CD34+c-kit- cells generated colony-forming cells (CFCs) after up to 9 weeks of coculture. Telomerase activity was consistently low in CD34+CD38- and CD34+c-kit- cells compared to CD38+ or c-kit(high or low) cells, suggesting that CD34+CD38- or c-kit- cells are likely to be more quiescent. These results suggest that the CD34+CD38- and CD34+c-kit- cell populations are primitive stem/progenitor cells, and that the telomerase activity of these cells correlates with their proliferative capacity as well as their stage of differentiation.

Development of a platform to evaluate and limit in-stent restenosis.

The objective of this work was to develop a platform to evaluate and deliver putative therapeutic agents for in-stent restenosis. Arterial stenting is applied in more than 60% of balloon angioplasties for treating cardiovascular disease. However, stented arteries encounter accelerated rates of restenosis. No prior platform has allowed evaluation or local management of in-stent restenosis without perturbing the very system being examined. A stainless steel, balloon-expandable stent was modified to serve as an ablumenal drug delivery platform. Several combinations of bioerodible polymer microspheres and gels were evaluated for channel retention under in vitro flow and in vivo conditions. A stent-anchored hybrid system prevented material embolization under all conditions. Unlike prior platforms, these stents do not alter local inflammation or in-stent plaque formation relative to conventional Palmaz-Schatz stents after in vivo deployment. The system also proved sensitive enough to detect plaque reduction with an antirestenotic agent. We conclude that a platform to evaluate and deliver therapeutic agents for in-stent restenosis has been achieved.

[Percutaneous hot ethanol injection therapy (PHEIT) for hepatocellular carcinoma].

Percutaneous ethanol injection therapy (PEIT) is widely performed as a local treatment for hepatocellular carcinoma (HCC). However, PEIT must be repeated because only a limited amount of ethanol can be injected in a single treatment. In addition, PEIT is only indicated for up to 3 tumors of 30 mm or less in diameter. We therefore invented a percutaneous hot ethanol injection therapy (PHEIT) to enhance the antitumor effect of PEIT. For this, we developed a liquid-heating and injection device to heat the liquid to the intended temperature and inject it from the tip of the needle safely and accurately using a puncture technique under ultrasonographic guidance. An experiment on normal rat livers demonstrated that injected ethanol at temperatures over 60 degrees C enhances the necrotizing effect on tissue. The technique was clinically applied to patients with HCC using ethanol heated to 60 degrees C or 70 degrees C. The total volume of the ethanol injection was smaller in PHEIT than in PEIT, and the frequency of injection was lower in PHEIT than in PEIT, while the necrotic area obtained by a single treatment was larger with PHEIT than with PEIT. PHEIT is a potential radical treatment for patients with multiple hepatoma with 4 more lesions or those with large hepatomas exceeding 30 mm in diameter that do not respond to PEIT.

Simultaneous signalling through c-mpl, c-kit and CXCR4 enhances the proliferation and differentiation of human megakaryocyte progenitors: possible roles of the PI3-K, PKC and MAPK pathways.

We assessed the effect of signalling through CXCR4 on the proliferation and differentiation of human megakaryocytic progenitor cells (CFU-Meg) in the presence or absence of stem cell factor (SCF) and/or thrombopoietin (TPO), using peripheral blood-derived CD34(+)IL-6R(-) cells as a target. TPO alone induced a significant number of CFU-Meg colonies. Although stromal cell-derived factor-1 (SDF-1) or SCF alone did not support CFU-Meg colony formation, these factors had a synergistic effect on CFU-Meg colony formation in the presence of TPO. The combination of SDF-1, SCF and TPO induced twice as many CFU-Meg colonies as TPO alone. To investigate the mechanism of this synergistic action, we examined the effects of various protein kinase inhibitors on CFU-Meg colony formation. LY294002 and GF109203X (inhibitors of PI3-K and PKC respectively) completely or partially inhibited this synergistic action. In contrast, a MEK inhibitor (PD98059) did not inhibit CFU-Meg colony formation. It significantly increased the higher ploidy classes (16N to 64N) of megakaryocytes supported by TPO, TPO + SCF, TPO + SDF-1, and TPO + SCF + SDF-1, whereas it abolished the effect of SDF-1 on the increase of higher ploidy classes of megakaryocytes supported by TPO. These results suggest that MAPK may negatively or positively regulate the nuclear maturation of megakaryocytes, known as endomitosis. In the presence of PD98059, proplatelet formation (PPF) was significantly augmented, suggesting that the MAPK pathway may also inhibit the initiation of PPF. In conclusion, simultaneous activation of three signals through c-mpl, c-kit and CXCR4 can induce the in vitro proliferation and differentiation of CFU-Meg, and SDF-1 is a potentiator of human megakaryocytopoiesis.

A case of interstitial pneumonitis associated with natural alpha-interferon therapy for myelofibrosis.

A 55-year-old man with myelofibrosis was treated with natural alpha-interferon with a good hematologic response. Initially, he had anemia, leukocytosis, thrombocytosis and hepatospleomegaly. A bone marrow biopsy showed replacement with fibrosis with an increase in megakaryocytes. Natural alpha-interferon (alpha-IFN) was started at a dose of 3 x 10(6) units/day. The leukocyte and platelet counts gradually normalized, and the liver and spleen decreased in size. However, the patient complained of a dry cough and dyspnea on the 61st treatment day, when the accumulated dose of alpha-IFN treatment had reached 1.8 x 10(8) units. He subsequently developed acute respiratory failure (PaO2 < 60 mm Hg) with bilateral lung infiltrations, suggesting the occurrence of interstitial pneumonitis associated with alpha-interferon therapy. Immediately, the alpha-interferon was discontinued and high-dose methylprednisolone (1.5 g/day) was administered for 3 days. This treatment was followed by oral prednisone therapy. Steroid therapy brought about gradual improvement as suggested by a repeat radiograph. Since high levels of fibrogenic cytokines, such as PDGF and TGF-beta, have been reported in patients with myelofibrosis, it is necessary to pay attention to interstitial pneumonia as a complication in alpha-IFN therapy for myelofibrosis.

Hepatocellular carcinoma: involvement of the internal mammary artery.

PURPOSE: To investigate factors related to the development of internal mammary arteries (IMAs) as feeding arteries of hepatocellular carcinomas (HCCs). MATERIALS AND METHODS: In 30 patients with HCC located in ventral hepatic areas directly beneath the diaphragm, bilateral internal mammary arteriography was performed to explore involvement of the IMA with HCC. The number of previous transcatheter arterial embolizations (TAEs), tumor size, time from initial TAE to IMA angiography, inferior phrenic artery (IPA) involvement with tumor, presence of hepatic artery occlusion, and use of other treatments were compared in groups with and without involvement of the IMA. RESULTS: The group with IMA involvement included 10 patients; the group without involvement, 20 patients. TAE had been performed two to 12 times in the group with involvement and zero to six times in the group without involvement (P =.01). Mean tumor sizes in these two groups were 5.1 and 6.0 cm, respectively; hepatic artery occlusion was noted in nine and zero patients (P =.01) in the two groups. The time from initial TAE to IMA angiography ranged from 3 to 53 months (median, 31.5 months) and from zero to 89 months (median, 0 months) (P =.01). IPA involvement was observed in seven and four patients (P =.015). CONCLUSION: These results strongly suggest that, regardless of tumor size, when HCCs are located in the ventral hepatic areas directly beneath the diaphragm, the IMAs serve as feeding arteries in patients with hepatic artery occlusion caused by repeated TAE.

Haematopoietic action of flt3 ligand on cord blood-derived CD34-positive cells expressing different levels of flt3 or c-kit tyrosine kinase receptor: comparison with stem cell factor.

We compared the effect of human flt3 ligand (FL) and stem cell factor (SCF) on cord blood (CB)-derived CD34+ cells expressing different levels of flt3 or c-kit tyrosine kinase (TK) receptor in clonal cell culture. The c-kit receptor was expressed by 58.5+/-16.7% of CB CD34+ cells (n=19), in which c-kit(high), c-kit(low) and c-kit cell populations could be identified. In contrast, the flt3 receptor (FR) was weakly expressed on 58.6+/-8.3% (n=9) of CB CD34+ cells. FL+erythropoietin (Epo) failed to support erythroid burst (BFU-E) formation by any subpopulation of CD34+ cells. However, SCF + Epo supported BFU-E and erythrocyte-containing mixed (CFU-mix) colony formation from all subpopulations. Interestingly, FL markedly augmented CFU-mix colony formation supported by interleukin (IL)-3 + Epo when CD34+c-kit(low) or CD34+FR+ cells were used as the target. On the other hand, SCF significantly enhanced CFU-mix colony formation supported by IL-3 + Epo when CD34+c-kit(high) or low and CD34+FR+ cells were used. The replating potential of CFU-mix supported by IL-3 + Epo+ FL was greater when CD34+c-kit(low) or CD34+FR+ cells were used. When the CD34+c-kit(low) cells were used, the number of lineages expressed in secondary cultures of CFU-mix colonies derived from primary cultures containing IL-3 + Epo + FL or SCF was significantly larger than when the primary cultures contained IL-3 + Epo. Furthermore, the number of long-term culture-initiating cells found in CD34+FR+ cells was larger than that in FR cells. CB-derived CD34+c-kit(low) cells represent a less mature population than c-kit(high) cells, as reported previously. Therefore, these results indicate that both FL and SCF can act on primitive multipotential progenitors. However, it is still uncertain whether CB-derived CD34+FR+ cells are less mature than CD34+FR- cells.

[Gore-tex covering of ultraflex stent and its usefulness for malignant esophageal stenosis: preliminary clinical results].

Malignant esophageal stenoses develop esophagorespiratory fistulae or perforations so frequently that esophageal stents must be required. We devised a covered stent with a thin Gore-Tex sheet and a nitinol stent system, Ultraflex, without increasing the size of the introducer assembly. Nine patients with malignant esophageal stenoses, including seven patients with perforation or fistulation, were carefully treated with the covered stent. All stent deployments were successfully carried out under fluoroscopic guidance. The average time required for full self-expansion was two weeks. The average grade of dysphagia improved from 3.7 to 1.2. Clinical symptoms due to esophagorespiratory fistulae were improved in three of four patients. No clinical or technical complications, such as migration, were observed, but a fistula developed at the bare site of the stent. This covering method for the flexible stent was simple and safe, and was considered to be useful in the treatment of malignant esophageal stenosis.

Signal through gp130 activated by soluble interleukin (IL)-6 receptor (R) and IL-6 or IL-6R/IL-6 fusion protein enhances ex vivo expansion of human peripheral blood-derived hematopoietic progenitors.

This study was designed to investigate the effects of a combination of soluble interleukin (sIL)-6 receptor (R) and IL-6 on the ex vivo expansion of human peripheral blood (PB)-derived hematopoietic progenitor cells in a short-term serum-free liquid suspension culture system, using PB-derived CD34(+)IL-6R(+/-) cells as a target. In combination with stem cell factor (SCF), IL-3, and sIL-6R/IL-6, the expansion efficiency (EE) for granulocyte/macrophage colony-forming unit (CFU-GM) reached a peak level on day 10 of incubation. On the other hand, the EE for erythroid burst (BFU-E) and mixed colony-forming unit (CFU-Mix) reached a peak level on day 7 of incubation. Among the cytokine combinations tested, SCF + IL-3 + sIL-6R/IL-6 + flt3 ligand (FL) most effectively expanded CFU-GM and CFU-Mix. The maximum EEs for CFU-GM and CFU-Mix were 208-fold and 42-fold, respectively. While the EE for BFU-E was 70-90-fold in the presence of SCF + IL-3 + sIL-6R/IL-6, FL significantly augmented the EE for CFU-GM and CFU-Mix. In contrast, thrombopoietin (TPO) significantly augmented the EE for CFU-Mix. Interestingly, in combination with IL-3 and SCF, newly generated IL-6R/IL-6 fusion protein (FP) expanded PB-derived BFU-E and CFU-Mix twice more effectively than a combination of sIL-6R and IL-6. These results demonstrated that human PB-derived committed progenitors were effectively expanded in vitro using sIL-6R/IL-6 or FP, in combination with IL-3, SCF and/or FL or TPO, and that FP may transduce a stronger intracellular signal than a combination of sIL-6R and IL-6.

Interleukin 6 receptor expression by human cord blood- or peripheral blood-derived primitive haematopoietic progenitors implies acquisition of different functional properties.

The significance of interleukin 6 receptor (IL-6R) expression by cord blood (CB)- and peripheral blood (PB)-derived primitive haematopoietic progenitors was investigated. IL-6R was preferentially expressed by PB-derived myeloid progenitors. Most PB-derived erythroid bursts (BFU-E) and mixed colony-forming cells (CFU-Mix) did not express this receptor. However, CB-derived primitive progenitor cells possessed multipotentiality, irrespective of IL-6R expression. Interestingly, the long-term culture-initiating cell (LTC-IC) population was enriched in PB-derived CD34+ IL-6R+ cells, but the extended LTC-IC (ELTC-IC) population, which represents a less mature class of haematopoietic progenitors, seemed to be equally distributed in the IL-6R+ and IL-6R- cell populations. In contrast, the number of LTC-ICs and ELTC-ICs was similar in CB-derived CD34+ IL-6R+ or IL-6R- cells. It is noteworthy that the number of LTC-ICs and ELTC-ICs in CB-derived CD34+ cells was markedly higher than that in PB-derived CD34+ cells regardless of IL-6R expression. Telomerase activity was consistently lower in PB-derived CD34+ IL-6R- cells than in CD34+ IL-6R+ cells. In contrast, telomerase activity was similar in CB-derived CD34+ IL-6R+ or IL-6R- cells. The pattern of telomerase induction upon cytokine stimulation differed between CB- and PB-derived CD34+ IL-6R+ or IL-6R- cells. However, overall telomerase activity per dish was well correlated with the proliferative potential of both cell populations, suggesting that induction of telomerase plays an important role in the escape from replicative senescence of primitive haematopoietic progenitors. Collectively, these results suggest that CB-derived primitive progenitors are less mature than PB-derived progenitors and that the expression of IL-6R by primitive haematopoietic progenitors may have different implications for PB- and CB-derived CD34+ cells.

Analysis of hepatocellular carcinoma fed by internal thoracic artery

Purpose: The purpose of this study was to elucidate the clinical features of hepatocellular carcinoma (HCC) fed by the internal thoracic artery (ITA). Methods: In seven patients HCC fed by the ITA was confirmed by digital subtraction angiography. The number of previous transcatheter arterial embolization (TAE), the period from the first TAE to TAE of the ITA, tumor location, tumor size, and occlusion of the hepatic artery (HA) and other collateral vessels were explored in each case. Results: The HCCs were located in S4 of the liver (n = 5) and in S8 (n = 1) and were fed by the right ITA and one nodule in S2-3 was fed by the left ITA. Tumor size was 3-10 cm. The number of previous TAE of the HA ranged from 2 to 12. The period from the first TAE to TAE of the ITA was 3-53 months. Angiography of these patients showed occlusion of the HA in six cases, and of the extrahepatic collaterals including the inferior phrenic artery (IPA) in five cases, intercostal artery (ICA) in one case, and epicholedocal artery (EPA) in one case. Conclusion: The ITA often supplies HCC located in the anterior superior region of the liver under the diaphragm; there can be long-term survival with repeated TAE and occlusion of HA.