Safety and efficacy of autologous bone marrow stem cell transplantation through hepatic artery for the treatment of chronic liver failure: a preliminary study.

This study was performed to determine the safety and tolerability of injecting autologous bone marrow stem cells (BMC) (CD34+) into four patients with liver insufficiency. The study was based on the hypothesis that the CD34+ cell population in granulocyte colony stimulating factor (G-CSF) mobilized blood and autologous bone marrow contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated the CD34+ stem cell population from the bone marrow. The potential of the BMC to differentiate into hepatocytes and other cell lineages has already been reported. Several reports have also demonstrated the plasticity of hematopoietic stem cells to differentiate into hepatocytes. Recently Sakaida demonstrated reduction in fibrosis in chemically induced liver cirrhosis following BMC transplantation. From a therapeutic point of view, chronic liver cirrhosis is one of the targets for BMC transplantation. In this condition, there is excessive deposition of extracellular matrix and hepatocyte necrosis. Encouraged by this evidence that the CD34+ cell population contains cells with the potential to form hepatocyte-like elements, four patients with liver insufficiency were given G-CSF to mobilize stem cells. CD34+ cells (0.1 x 10(8)) were injected into the hepatic artery. No complications or specific side effects related to the procedure were observed; four patients showed improvements in serum albumin, bilirubin and ALT after one month from the cell infusion.

Destruction and creation of spatial tuning by disinhibition: GABA(A) blockade of prefrontal cortical neurons engaged by working memory.

Local circuit neurons in the dorsolateral prefrontal cortex (dPFC) of monkeys have been implicated in the cellular basis of working memory. To further elucidate the role of inhibition in spatial tuning, we iontophoresed bicuculline methiodide (BMI) onto functionally characterized neurons in the dPFC of monkeys performing an oculomotor delayed response task. This GABA(A) blockade revealed that both putative interneurons and pyramidal cells possess significant inhibitory tone in the awake, behaving monkey. In addition, BMI application primarily resulted in the loss of previously extant spatial tuning in both cell types through reduction of both isodirectional and cross-directional inhibition. This tuning loss occurred in both the sensorimotor and mnemonic phases of the task, although the delay activity of prefrontal neurons appeared to be particularly affected. Finally, application of BMI also created significant spatial tuning in a sizable minority of units that were untuned in the control condition. Visual field analysis of such tuning suggests that it is likely caused by the unmasking of normally suppressed spatially tuned excitatory input. These findings provide the first direct evidence of directional inhibitory modulation of pyramidal cell and interneuron firing in both the mnemonic and sensorimotor phases of the working memory process, and they implicate a further role for GABAergic interneurons in the construction of spatial tuning in prefrontal cortex.

A new method for the growth of myeloid progenitor cells on nitrocellulose paper.

A new method has been developed by which human myeloid progenitor cells can be grown on nitrocellulose paper. The method uses human placental conditioned medium for colony-stimulating activity in an agar layer while the cells grow on the overlying nitrocellulose paper in compact colonies containing granulocytic and macrophage cells. The importance of this method is discussed in the light of its usefulness in carrying out molecular studies on differentiating hematopoietic cells.

Growth factors in chronic myelogenous leukemia.

Transforming growth factors (TGFs) are implicated in malignancy, therefore qualitative and quantitative differences of these growth factors in chronic myelogenous leukemia (CML) in chronic phase has been investigated in this report. Induction of anchorage independent growth of BALB/c 3T3 and NRK-49F fibroblasts was used as an assay to detect TGF-beta activity in sera, serum free leukocyte and stromal conditioned medium of CML patients as well as normal subjects. The data shows that enhanced levels of transforming growth factor (type beta like activity) are detectable in the sera of chronic myelogenous leukemia patients. We believe that the enhanced levels of TGF-beta type activity may have a role in myeloid hyperplasia characteristic of CML patients in chronic phase of the disease.

Inhibition of lung homing of B16F10 by pentoxifylline, a microfilament depolymerizing agent.

B16 melanoma has proved to be an ideal model for investigating metastasis. The parental B16F1 line grows as a localized subcutaneous tumor in C57BL/6 or DBA/2 mice. However, by means of successive intravenous transplantation, a subline B16F10 has been established. This shows preponderant lung homing when transplanted by intravenous route into C57BL/6 mice. In this paper we have shown that pentoxifylline (PTX; Hoechst), a microfilament depolymerising agent can inhibit significantly this lung homing of B16F10 cells. It also had a marginal inhibitory effect on subcutaneous tumor growth.

A mitogenic factor from rat muscle.

A growth-promoting polypeptide has been purified about 1950-fold from rat abdominal muscle by gel chromatography, isoelectric-focusing (IEF) and reverse phase high pressure liquid chromatographic (RP-HPLC) techniques. This factor is an acidic protein with an isoelectric point (pI) of 3.4 and a mol. wt of 12 kDa when run on SDS-polyacrylamide gels under reducing conditions. It is acid and heat-stable but is sensitive to reducing agent and protease action. It stimulates [3H]thymidine incorporation in NRK-49F and NIH 3T3 cells and induces soft agar colony formation of NIH 3T3 cells. The muscle-derived mitogen appears to differ from other known growth factors (GFs) in its physico-chemical properties.

Disruption of stromal niches by diffusible factors in the conditioned media of leukemic cell-lines and sera of chronic myelogenous leukemia patients.

The bone-marrow microenvironment has a decisive role in maintaining haemopoietic stem cells and regulating their differentiation. In diseases of the haemopoietic system, viz. anaemias and leukemias, the microenvironment has been shown to play a key role. In this paper we show that leukemic cell conditioned medium and sera from CML patients, interact with the in vitro bone-marrow environment and bring about changes which affect the maintenance of normal stem cells.

Evolving surgical management for ventricular septal defect, pulmonary atresia, and major aortopulmonary collateral arteries.

BACKGROUND: The purpose of this study was to evaluate the results of various surgical modalities that have been evolving for the treatment of ventricular septal defect, pulmonary atresia, and major aortopulmonary collateral arteries. METHODS: From 1993 to May 1997, 14 patients (group 1) were treated with staged unifocalization through thoracotomies and final repair by midsternotomy. From June 1997 to February 1998, 10 patients (group 2) were treated with midsternotomy, single-stage complete unifocalization, and repair. RESULTS: In group 1, 14 patients had 21 procedures (1.5 procedures per patient), of which 3 patients (21%) had final correction. There were two deaths (14%). One patient died of blocked shunt. Another patient who had aneurysmal dilation of homograft tubes that were used for unifocalization died after final repair because of low cardiac output. In group 2, 10 patients had ten surgical procedures for complete unifocalization and 9 of 10 (90%) of them achieved final correction. One patient with low cardiac output in whom we did not close the ventricular septal defect died (10%) of suprasystemic right ventricular pressure. CONCLUSION: In single-stage complete unifocalization, more patients had final correction. It reduces the number of operations and hospitalization and hence is more cost effective than multistaged procedures.

Role of limited posterior thoracotomy for open-heart surgery in the current era.

BACKGROUND: The earliest open-heart operations were performed employing the thoracotomy approach. Over the years, median sternotomy has become the routine way of approaching the heart. However, lately there has been progressive enthusiasm in minimally invasive techniques for accessing the heart. We present our technique of correction of congenital heart defects employing the limited posterior thoracotomy approach. METHODS: From June 1997 to April 1998, 27 patients underwent correction for various intracardiac defects without any mortality. There were 19 ostium secundum defects, with or without other associated anomalies. There were six sinus venosus defects with partial anomalous pulmonary venous connections. Two patients had perimembranous ventricular septal defects, while 2 patients had partial atrioventricular defects. In 2 other patients, pulmonary stenosis was repaired, using pulmonary valvotomy in 1 patient, whereas the other patient required short transannular patch. RESULTS: The median age was 7 years and the median weight was 20 kg. The median skin-to-skin time was 260 minutes. The median bypass time was 63.25 minutes and the median cross-clamp time was 35.0 minutes. All the patients were extubated within 12 hours following surgery and the median ICU stay was 24 hours. Three patients required blood transfusions in the ICU for significant blood loss and the mean chest drainage was 85 cc per 24 hours. None of the patients had phrenic nerve palsies. None of the patients required additional analgesics other than routine ibuprofen or ketorolac tromethamine. Short-term follow-up revealed no functional or physical disability of the thoracic wall and the right arm. All who underwent surgery with this approach were happy with the limited visibility of their scars. CONCLUSIONS: Limited posterior thoracotomy offers a viable alternative for midsternotomy and submammary thoracotomy. It has the advantage of a scar in the back that does not impede the future growth of the breast tissue and the pectoralis major. Our approach does not need any new instruments and hence no contraptions are necessary to perform the operation with this approach. Our results have shown satisfactory short-term results and better cosmesis.

Terminal differentiation of human leukemic blasts in response to 12-0-tetradecanoyl-13-phorbol acetate (TPA).

Phorbol ester, 12-0-Tetradecanoyl-13-Phorbol-Acetate (TPA), induces a terminal macrophage-like differentiation of cells from human acute myelogenous leukemia cell lines. We report here that blastoid cells obtained from acute nonlymphocytic leukemia (M1-M2) undergo differentiation-related changes characteristic of macrophage lineage after exposure to TPA. Blast cells from a patient with ANLL-M1-M2 underwent morphological, functional and histochemical changes after treatment with 1 x 10(-7) and 1 x 10(-8) M TPA. The changes included adhesion to the plastic substrate, 2-4 fold increase in the number of NBT positive cells and an increase in the number of alpha-naphthyl-acetate esterase (alpha-NAE) positive cells. These differentiation changes after treatment with TPA were followed by decrease in proliferative index and G1 cells containing high RNA as estimated by flow cytometry. Of the thirteen cases of undifferentiated or unclassified leukemias studied, two failed to respond to TPA. These data suggest that leukemic blasts retain their ability to express a variety of differentiated functions on induction by TPA. Our data gives evidence suggesting that the "switch" into the differentiation pathways occurred after inhibition of proliferation and reduction in the percentage of G1 high RNA containing cells.

Sterically stabilized etoposide liposomes: evaluation of antimetastatic activity and its potentiation by combination with sterically stabilized pentoxifylline liposomes in mice.

Cytotoxic activity of chemotherapeutic agents can be enhanced by site-specific delivery or by combination with other less toxic agents. In the present study, enhancement in the antimetastatic activity of etoposide (ETP) by encapsulation in sterically stabilized liposomes was evaluated in the murine experimental B16F10 melanoma model. Further, potentiation of its antimetastatic activity by combination with pentoxifylline (PTX) solution or sterically stabilized PTX liposomes was evaluated in the same animal model. Upon intravenous administration, ETP solution and ETP liposomes inhibited pulmonary tumor nodule formation in a dose-dependent manner. Encapsulation of ETP in liposomes resulted in significant enhancement in its antimetastatic activity at doses of 0.5 mg/kg and 0.75 mg/kg as compared to ETP solution at similar doses. In combination therapy, the effect of sequence of administration of the drugs, ETP and PTX, was evaluated. Enhancement of antimetastatic activity of ETP solution when used in combination with PTX solution was effected by the sequence in which the solutions were administered. However, a combination of ETP liposomes and PTX liposomes led to potentiation of antimetastatic activity in a sequence-independent manner. The results indicate that antimetastatic activity of ETP is significantly enhanced by encapsulation in liposomes. Administration of ETP liposomes with PTX liposomes further potentiated the activity, suggesting the usefulness of this combination in clinical practice for reducing the dose-limiting toxic effects of ETP.

Effects of niosomal cisplatin and combination of the same with theophylline and with activated macrophages in murine B16F10 melanoma model.

The use of cisplatin is limited due to its certain toxic effects. In the present study niosomes of cisplatin by using span 60 and cholesterol were prepared and investigated for antimetastatic activity in experimental metastatic model of B16F10 melanoma. Theophylline and its combination effect with free cisplatin and niosomal cisplatin were also carried out in the same model. The effect of treatment with activated macrophages alone and in combination with cisplatin, theophylline and niosomal cisplatin was also observed. Treatment with niosomal cisplatin (1 mg/kg) and combination of the same with theophylline (15 mg/kg) showed significant reduction in the number of lung nodules as compared to untreated control as well as with free cisplatin (1 mg/kg). The treatment with activated macrophages (activated by using Muramyl dipeptide) significantly reduced the secondary growth of tumor in lung. Niosomal cisplatin showed a significant protection against weight loss and bone marrow toxicity as compared to free cisplatin. These results suggest that cisplatin encapsulated in niosomes has significant antimetastatic activity and reduced toxicities than that of free cisplatin. However theophylline failed to show antimetastatic effect alone or in combination with cisplatin or with activated macrophages.

New spectrophotometric method for the determination of some drugs with iodine and wool fast blue BL.

A simple, sensitive and selective method is proposed for the spectrophotometric determination of drugs, viz. ampicillin, penicillin V, amoxycillin, cloxacillin, cefadroxil, ceftezoxime, griseofulvin, streptomycin, nicoumalone and acebutolol HCl, based on their reactivity with iodine. The method involves the addition of excess iodine of known concentration to the drug in the presence of NaOH and the unreacted iodine is determined by the measurement of the decrease in the absorbance of the dye wool fast blue BL (lambda(max)=540 nm) which was found to be the most suitable of several dyes tested. This method was applied for the determination of drug contents in pharmaceutical formulations and enabled the determination of the drugs in microgram quantities (0.8-9.6 mug ml(-1)). No interferences were observed from excipients and the validity of the method was tested against reference methods.

Mast cells in leprosy skin lesions.

The density and distribution of mast cells was assessed in skin biopsies of 118 untreated leprosy cases and 20 healthy individuals taken as controls. Mast cells were present in only small numbers in the skin biopsies of healthy individuals. Significantly higher mast cell counts were obtained in the skin lesions of indeterminate leprosy (P < 0.01). The mast cell count in the tuberculoid group was significantly lower than that in the lepromatous group (P < 0.05). The lepromatous group also showed increased mast cell degranulation and altered morphology. The mast cell distribution in the skin biopsies of the two groups was, however, similar. The mast cell count in leprosy is probably determined by the pattern of cytokines released by the T lymphocytes. However, the influence of mast cells on the outcome of the disease needs to be evaluated further.

Erythrocyte depletion of human umbilical cord blood using dextran sedimentation.

We report on the results of a study using high molecular weight dextran for depletion of red blood cells (RBCs) from cord blood. Our technique achieved efficient RBC depletion by sedimentation without a significant loss in haemopoietic stem cells. Cord blood units were fractionated for erythrocyte depletion by unit gravity sedimentation in 3 per cent high molecular weight dextran. Dextran sedimentation enabled recovery of more than 80 per cent of the total nucleated cells present and 100 per cent mononuclear cell (MNC) recovery as compared to unfractionated cord blood. A four-fold increase in the colony forming unit-granulocyte macrophage (CFU-GM) number per 2 x 10(5) cells was observed after dextran treatment suggesting that this step also resulted in the enrichment of stem cells.

Umbilical cord blood as a source of CD34 positive haematopoietic stem cells.

In this paper we have used a monoclonal antibody to CD34 an antigen expressed solely on stem cells, and stem cell colony assays to show that umbilical cord blood has nearly the same number of functional stem cells as compared to normal bone-marrow. The number of CD34+ve cells in cord blood being 2 to 2.7 per cent, whereas bone-marrow had 3 to 3.5 per cent. The multi-potent colony forming cells (CFU-GEMM) were 60 +/- 18 in cord blood per 2 x 10(5) mononuclear cells (MNCs), whereas normal bone-marrow had 70 +/- 10 per 2 x 10(5) MNCs. Enrichment of these stem cells on Percoll gradients was successful for normal bone-marrow but not for cord blood.

Characterization of a pan-lymphocyte monoclonal antibody useful in differentiating leukemic from normal myeloid progenitors and monocytes from macrophages.

A monoclonal antibody (McAb) designated 3A2 that recognizes a 51 kDa epitope having surface density of 37 x 10(8) per MOLT-4 cells is described. This epitope appears to be expressed on (i) lymphocytes at all stages of differentiation; (ii) leukemic myeloid progenitors; (iii) peripheral blood monocytes (MO). The epitope is specifically absent from normal myeloid progenitors and macrophages. The McAb may, therefore, be useful in studying myeloid lineage leukemias and, as a marker for monocyte to macrophage (MO + MAC) differentiation.

Enhancement of anti-metastatic activity of pentoxifylline by encapsulation in conventional liposomes and sterically stabilized liposomes in murine experimental B16F10 melanoma model.

Pentoxifylline has been shown to exhibit anti-metastatic activity by inhibiting homing of B16F10 melanoma cells in the murine experimental metastasis model. In this study, the effect of encapsulation of pentoxifylline in conventional and sterically stabilized liposomes on its anti-metastatic activity in the murine experimental metastasis model was investigated. After a single intravenous dose (10, 20 or 40 mg kg(-1)), pentoxifylline solution, as well as conventional pentoxifylline liposomes, significantly reduced the number of pulmonary nodules compared with the untreated control group. Conventional pentoxifylline liposomes showed significantly higher inhibition (69%) of pulmonary tumour nodule formation at a dose of 20mg kg(-1) as compared with pentoxifylline solution (49%) at the same dose. Encapsulation of pentoxifylline in sterically stabilized liposomes prepared by incorporation of monomethoxypolyethyleneglycol (5000)-cholesteryl ester further enhanced the inhibition of pulmonary nodule formation (77%) at a dose of 20 mg kg(-1) as compared with conventional pentoxifylline liposomes. Overall, the results suggest that encapsulation of pentoxifylline in conventional liposomes enhanced its anti-metastatic activity. Steric stabilization of pentoxifylline liposomes also resulted in a two-fold increase in anti-metastatic activity (at dose of 10 mg kg(-1)) as compared with conventional liposomes.

Effect of Caffeine, a xanthine derivative, in the inhibition of experimental lung metastasis induced by B16F10 melanoma cells.

Caffeine, a methyl xanthine derivative, was studied to assess the effect on B16F10 melanoma induced experimental metastasis. Caffeine was administered at a dose of 100 and 50 mg/kg body weight by both routes, to tumour bearing animals. Solid tumour reduction studies with Caffeine showed a significant reduction in tumour volume for 100 mg/kg dose by both oral and i.p. routes. The Caffeine treated metastatic tumour bearing animals significantly (p<0.001) inhibited lung tumour nodules. Serum sialic acid levels and lung hydroxyproline contents in the treated groups were significantly (p<0.001) low, when compared with the untreated control animals. In the present study, our results suggest that Caffeine inhibits solid tumour development and pulmonary experimental metastasis induced by B16F10 melanoma cells, in murine model.

Antisense RNA therapy for CML--an hypothesis.

CML, a myeloproliferative clonal disorder of myeloid stem cells, is characterized by the consistent presence of a bcr-c-abl fusion gene which is formed as a result of a translocation of the c-abl gene from chromosome 9 to downstream of the bcr gene on chromosome 22 (ph'). Current approaches to the treatment of CML are chemotherapy (conventional or aggressive with immuno-modulators) and bone marrow transplantation (BMT). Neither of the above treatment modalities results in long-term remission or cure. Hence, an alternative approach which aims at correcting the genetic defect should be considered. Taking advantage of the consistent abnormal presence of the bcr-c-abl gene in the treated and untreated CML patients at all stages, a gene therapy at the level of blocking mRNA might be considered. Such an antisense RNA therapy should include removal of patient's bone marrow, administration of the gene for constitutive expression of an antisense RNA for the bcr-c-abl fusion gene into the myeloid stem cells and reinjecting the engineered marrow into the patient. Such an approach, comparable to autologous BMT, will have the advantages of absence of graft rejection and possibility of 100% remission. The possible nature of the gene construct for such an antisense RNA therapy is discussed.