Very small embryonic-like stem cells with maximum regenerative potential get discarded during cord blood banking and bone marrow processing for autologous stem cell therapy.

Very small embryonic-like stem cells (VSELs) are possibly lost during cord blood banking and bone marrow (BM) processing for autologus stem cell therapy mainly because of their small size. The present study was conducted on human umbilical cord blood (UCB, n=6) and discarded red blood cells (RBC) fraction obtained after separation of mononuclear cells from human BM (n=6), to test this hypothesis. The results show that VSELs, which are pluripotent stem cells with maximum regenerative potential, settle along with the RBCs during Ficoll-Hypaque density separation. These cells are very small in size (3-5 µm), have high nucleo-cytoplasmic ratio, and express nuclear Oct-4, cell surface protein SSEA-4, and other pluripotent markers such as Nanog, Sox-2, Rex-1, and Tert as indicated by immunolocalization and quantitative polymerase chain reaction (Q-PCR) studies. Interestingly, a distinct population of slightly larger, round hematopoietic stem cells (HSCs) with cytoplasmic Oct-4 were detected in the "buffy" coat, which usually gets banked or used during autologus stem cell therapy. Immunohistochemical studies on the umbilical cord tissue (UCT) sections (n=3) showed the presence of nuclear Oct-4-positive VSELs and many fibroblast-like mesenchymal stem cells (MSCs) with cytoplasmic Oct-4. These VSELs with nuclear Oct-4, detected in UCB, UCT, and discarded RBC fraction obtained after BM processing, may persist throughout life, maintain tissue homeostasis, and undergo asymmetric cell division to self-renew as well as produce larger progenitor stem cells, viz. HSCs or MSCs, which follow differentiation trajectories depending on the somatic niche. Hence, it can be concluded that the true stem cells in adult body tissues are the VSELs, whereas the HSCs and MSCs are actually progenitor stem cells that arise by asymmetric cell division of VSELs. The results of the present study may help explain low efficacy reported during adult autologous stem cell trials, wherein unknowingly progenitor stem cells are injected rather than the pluripotent stem cells with maximum regenerative potential.

Transglutaminase-2 regulation by arecoline in gingival fibroblasts.

Transglutaminase-2 (TGM-2) stabilizes extracellular matrix (ECM) proteins by cross-linking and has been implicated in several fibrotic disorders. Arecoline present in betel quid has been proposed as one of the causative factors for oral submucous fibrosis (OSMF). Hence, we hypothesize that arecoline may regulate TGM-2 and may have a role in the pathogenesis of OSMF. The expression of TGM-2 was studied in OSMF tissues by real-time RT-PCR analysis, and significant overexpression was observed in most OSMF tissues (P=0.0112) compared with normal tissues. Arecoline induced TGM-2 mRNA and protein expression as well as TGM-2 activity in human gingival fibroblast cells. The addition of methocramine hemihydrate (M-2 muscarinic acetylcholine receptor selective antagonist) or 8'-bromo-cAMP abolished arecoline-mediated TGM-2 induction, suggesting a role for M-2 muscarinic acid receptor and a repressor role for cAMP. Our study provides evidence for TGM-2 overexpression in OSMF and its regulation by arecoline in oral fibroblasts.

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.

Obesity and obstetric anaesthesia.

The prevalence of obesity continues to increase despite preventive strategies. Obese parturients are at increased risk of having either concurrent medical problems or superimposed antenatal diseases such as pre-eclampsia and gestational diabetes. Moreover, they have a tendency to labour abnormally contributing to increased instrumental delivery and Caesarean section. Obesity is a risk factor for anaesthesia related maternal mortality. Morbidly obese women must be considered as high-risk and deserve an anaesthetic consultation during their antenatal care. The significant difficulty in administering epidural analgesia should not preclude their use in labour. A more liberalised use of regional techniques may be a means to further reduce anaesthesia-related maternal mortality in the obese population. The mother's life should not be jeopardised to save a compromised fetus. Prophylactic placement of an epidural catheter when not contraindicated in labouring morbidly obese women would potentially decrease anaesthetic and perinatal complications associated with attempts at emergency provision of regional or general anaesthesia. Early mobilisation, aggressive chest physiotherapy and adequate pain control are essential components of effective postoperative care.

Successful management of isolated ventricular inversion in an adult.

We report a case of isolated ventricular inversion in a 42-year-old woman. This rare congenital cardiac anomaly was corrected by an intraatrial baffle procedure. She also underwent left-sided double-chamber endocardial pacemaker implantation for postoperative tachycardia bradycardia syndrome.

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.

Ex vivo expansion of stem cells from umbilical cord blood: expression of cell adhesion molecules.

Expansion of stem cells from cord blood has been demonstrated to increase the numbers of CD34+ cells, CD34+ subsets, long-term culture-initiating cells, and severe combined immunodeficient mouse, repopulating cells. However, reports suggest that the ex vivo expanded population behaves differently than freshly isolated cells and shows a delayed or diminished engraftment. In this study, we investigated the effects of the cytokines flt3 ligand, stem cell factor, and thrombopoietin on expansion of CD34+ and CD34+/CD38- cells. In addition, we studied the expression of adhesion molecules, very late activation antigen-4 (VLA-4) and leukocyte function antigen-1 (LFA-1), on CD34+ cells from cord blood by flow cytometry. We also looked at the expression of an adhesion receptor, namely, vascular cell adhesion molecule-1 (VCAM-1) on bone marrow stromal cells by Western blot analysis after exposure to low dose gamma irradiation. After culturing for 7 days, increases in the absolute numbers of CD34+, CD34+/CD38-, CD34+/VLA-4+, and CD34+/LFA-1+ cells were 5.67 +/- 2.91 (mean +/- standard deviation) fold, 7.21 +/- 4.38 fold, 99.56 +/- 101.5 fold, and 101.39 +/- 83.25 fold, respectively. There was a transient upregulation in the expression levels of VCAM-1 on stromal cells, which peaked at 4 hours. Though there was an increase in the absolute numbers of CD34+ cells expressing the adhesion molecules, the expression levels (antigen density) of the adhesion molecules on the CD34+ cells remained unaffected.

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.

Operative outcome and intermediate term follow-up of neonatal Blalock-Taussig shunts.

BACKGROUND: The neonatal age group is considered to be one of the important risk factors for perioperative morbidity and mortality as well as poor long-term patency following Blalock-Taussig shunts. METHODS AND RESULTS: Out of a total of 190 patients who underwent Blalock-Taussig shunts in our institute between July 1998 and July 2000, 20 patients were aged less than 30 days and this neonatal cohort was studied retrospectively. The mean age was 18+/-11 days (range: 3-30 days). The mean weight of the babies was 3.1+/-0.7 kg, the smallest weighed 2.1 kg. The cardiac anatomy was tetralogy of Fallot with pulmonary atresia in 6, pulmonary atresia with intact ventricular septum in 3, tricuspid atresia in 5 and complex single ventricle physiology in the rest. All patients were deeply cyanotic and preoperative prostaglandin E1 was needed in 10 patients to ensure ductal patency and maintain oxygen saturations prior to the shunt operation. The mean hilar right and left pulmonary artery sizes were 3.99+/-0.44 mm and 3.69+/-0.79 mm, respectively. Three patients (15%) had significant stenosis at the site of duct insertion. The shunts were accomplished with 3.5 mm polytetrafluoroethylene grafts in 7 patients (35%) and 4 mm in the rest. The mean duration of mechanical ventilation was 2.0+/-2.83 days, one patient who developed bronchopneumonia needed prolonged ventilation for 14 days. The mean intensive care unit stay was 4.79+/-2.66 days. The mean hospital stay was 11.7+/-6.4 days. Five patients who developed sepsis stayed beyond 14 days. There were 3 deaths (immediate post-operative shock and possibly shunt malfunction in 1, bronchopneumonia in 1 and late shunt thrombosis at 3 months in 1). Two patients had late shunt block, one of those mentioned above and the other at 3 months secondary to infective endarteritis of the right pulmonary artery. All these infants received 4 mm grafts. All the 3.5 mm grafts were patent at follow-up. Seventeen patients were alive and well at follow-up (mean: 9 months, range: 3-21 months) with a mean resting systemic oxygen saturation of 77% (66%-95%). CONCLUSIONS: The overall shunt patency rate after neonatal Blalock-Taussig shunt is about 80% on intermediate term follow-up. A smaller graft size (3.5 mm) does not appear to be an incremental risk factor for shunt blockade and operative mortality.

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.

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.

Mesenteric panniculitis presenting as a huge retroperitoneal mass--a case report.

A rare case of mesenteric panniculitis occurring in a young patient and presenting as a huge retroperitoneal mass which was mistaken for malignancy, has been described.

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 Hepatogard--an indigenous formulation on dexamethasone induced antihealing effects in male albino rats.

Hepatogard, is a multi-ingredient phytopharmaceutical product containing crude powders of eleven plants. Its effect on the different parameters of wound healing was assessed alone and in the presence of dexamethasone. The parameters chosen for the study were the breaking strength of incised wound, breaking strength of granulation tissue and hydroxyproline content. The result showed that Hepatogard increased the breaking strength of granulation tissue but not of incised wound. It reversed the dexamethasone induced decrease in breaking strength in both incised wound and granulation tissue. Even though it had no effect of its own on hydroxyproline concentration, it reversed the dexamethasone induced decrease in the hydroxyproline content of granulation tissue. Thus, Hepatogard has the potential for antagonizing the antihealing effect of steroids in patients receiving steroid therapy.

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.

Isodirectional tuning of adjacent interneurons and pyramidal cells during working memory: evidence for microcolumnar organization in PFC.

Studies on the cellular mechanisms of working memory demonstrated that neurons in dorsolateral prefrontal cortex (dPFC) exhibit directionally tuned activity during an oculomotor delayed response. To determine the particular contributions of pyramidal cells and interneurons to spatial tuning in dPFC, we examined both individually and in pairs the tuning properties of regular-spiking (RS) and fast-spiking (FS) units that represent putative pyramidal cells and interneurons, respectively. Our main finding is that FS units possess spatially tuned sensory, motor, and delay activity (i. e., "memory fields") similar to those found in RS units. Furthermore, when recorded simultaneously at the same site, the majority of neighboring neurons, whether FS or RS, displayed isodirectional tuning, i.e., they shared very similar tuning angles for the sensory and delay phases of the task. As the trial entered the response phase of the task, many FS units shifted their direction of tuning and became cross-directional to adjacent RS units by the end of the trial. These results establish that a large part of inhibition in prefrontal cortex is spatially oriented rather than being untuned and simply regulating the threshold response of pyramidal cell output. Moreover, the isodirectional tuning between adjacent neurons supports a functional microcolumnar organization in dPFC for spatial memory fields similar to that found in other areas of cortex for sensory receptive fields.

Role of inhaled nitric oxide as a selective pulmonary vasodilator in pediatric cardiac surgical practice.

Our aim was to assess the role of inhaled nitric oxide (NO) therapy in post operative cases of congenital heart defects who developed pulmonary arterial hypertensive (PAH) crisis and had no response with conventional management. From February '95 to January '97, inhaled NO therapy was used in 21 children. Age ranged from 2 months to 9 years (mean 5.6 years) and duration of therapy ranged from 1 to 13 days. Of 21 patients, 17 responded well with 5-20 ppm while 4 did not. The preoperative mean pulmonary systolic pressure was 88 mm Hg against mean systemic pressure of 96 mm Hg. Post operatively, their PA pressure reduced to 62 mm Hg, with systemic pressure of 98 mm Hg. After using inhaled NO, PA pressure dropped to 24 mm Hg (mean systolic) (p < 0.007), after excluding the non responders. Of 4 non responders, two died due to irreversible pulmonary vascular disease and remaining two died due to residual defects. The study shows that inhaled NO is a selective pulmonary vasodilator, which is useful in postoperative PAH crisis and also reduces the transpulmonary gradient in single ventricle repair cases. It is safe and effective for prolonged use. It is very useful in Indian perspective, when more number of cases with congenital heart defects (CHD) along with severe PAH are encountered routinely.