An in vitro study of the impact of 4mT static magnetic field to modify the differentiation rate of rat bone marrow stem cells into primordial germ cells

This investigation was performed to evaluate the differentiation capacity and alteration in genes expression patterns during in vitro differentiation of bone marrow stem cells into primordial germ cells using static magnetic field (4 mT) and BMP-4 (25 ng/ml). The rate of differentiation was investigated using the Real Time-PCR method for tracing expression of differentiation markers (Oct-4, Nanog, C-Myc, Fragilis, Mvh and Stella). Then, immunocytochemical reaction was carried out for detection of marker proteins (Oct4 and Mvh). Increasing of the exposure time of the 4 mT SMF (24 and 48 h) and treatment time with 25 ng/ml BMP4 (48 and 96 h) indicated a marked decrease in expression of pluripotency genes (Oct-4, Nanog and C-Myc) and Oct4 protein and increase in primordial germ cell-specific genes (Fragilis, Mvh and Stella) and Mvh protein compared with the control group. Final results showed that in a synergistic manner, the combination of SMF with BMP4 exaggerates the differentiation potential of BMSCs to PGCs by activating the MAPK pathway and altering the Ca²⁺ concentration.     

Bone morphogenetic protein 2 promotes osteogenesis of bone marrow stromal cells in type 2 diabetic rats via the Wnt signaling pathway

Type 2 diabetes mellitus impairs osteogenesis in bone marrow stromal cells (BMSCs). Bone morphogenetic protein 2 (BMP2) has been extensively applied for bone defect restoration and has been shown to activate the Wnt signaling pathway. The objective of this study was to investigate the effects of BMP2 on the cell proliferation and osteogenesis of type 2 diabetic BMSCs in rats and explore whether BMP2 induced osteogenesis via the stimulation of Wnt signaling pathway. The cell experiments were divided into DM (diabetic BMSCs), BMP25 (induced with 25 ng/ml BMP2), BMP100 (induced with 100 ng/ml BMP2) and BMP25  + XAV groups. All cells with or without the different concentrations of BMP2 were cultured under the same experimental conditions. The in vitro results indicated that BMP2 enhanced cell proliferation by 130%–157% and osteogenic differentiation by approximately two-fold in type 2 diabetic BMSCs. The expression levels of β-catenin, cyclin D1, Runx2 and c-myc related to the Wnt signaling pathway were also upregulated from 180% to 212% in BMP2-induced type 2 diabetic rat BMSCs, while the level of GSK3β decreased to 43%. In BMP2-induced type 2 diabetic BMSCs with calcium phosphate cement (CPC) scaffolds for osteoblast study in vivo, the appearance of newly formed bone dramatically increased to 175% compared with type 2 diabetic BMSCs. These data demonstrated that BMP2 enhanced bone regeneration in diabetic BMSCs by stimulating the Wnt signaling pathway with the accumulation of β-catenin and the depressed expression of GSK3β. Diabetic BMSCs associated with BMP2 might be a potential tissue-engineered construct for bone defects in type 2 diabetes mellitus.     

Association of immune parameters with clinical outcome in stage III colon cancer: results of Southwest Oncology Group Protocol 9009

Levamisole (LMS), utilized in the adjuvant treatment of patients with stage III colon cancer, is immunomodulatory. To determine whether alterations in immune parameters before, during and after 12 months of 5FU/LMS therapy correlate with disease-free survival, 38 patients enrolled on Southwest Oncology Group (SWOG) protocol 8899 received extensive lymphocyte phenotypic analysis prior to therapy and 3, 6, 12 and 15 months after treatment initiation. The median follow-up of patients is 41 months. Significant increases in the proportion and total number of CD56⁺ natural killer cells were seen, starting at 3 months and continuing until 15 months (P < 0.001). Increases in the total numbers of cells expressing CD25 (interleukin-2 receptor), VLA4 and the combinations of CD4: CD45RA and CD4:CDw29 were not evident during therapy but were seen at 15 months (P < 0.05: CD25, CD4:CDw29, CD4:CD45RA; P < 0.001: VLA4). Low levels of CD8⁺ cells prior to treatment initiation and after 3 months of therapy correlated with early relapse within the first year of 5FU/LMS treatment. Patients who have remained disease-free (n = 22, median follow-up 45 months) demonstrated increases in the total numbers of CD8⁺, CD25⁺, CD56⁺, VLA4⁺, CD4: CDw29 and CD4:CD45RA cells, primarily at 15 months. In contrast, patients who relapsed had decreased numbers of CD8⁺, CD4:CDw29, CD4: CD45RA and VLA4⁺ cells and minimal increases in CD56⁺ and CD25⁺ cells. Statistically significant differences between the late-relapse group and the group remaining disease-free were seen for CD25⁺, CD4: CD45RA and CD4:CDw29 cells at the 15-month assay time (P = 0.0276, P = 0.0349, P = 0.0178 respectively). In conclusion, multiple alterations in lymphocyte phenotype, with increases in the proportion and total number of cells involved in cell-mediated immune responses, were seen during and especially following completion of therapy with 5FU/LMS. Many of these changes are significantly associated with clinical outcome and may be useful for risk stratification of stage III colon cancer patients following completion of adjuvant therapy. Received: 9 July 1999 / Accepted: 11 August 1999     

The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.     

Bone morphogenetic protein (BMP)-7 but not BMP-2 and BMP-4 improves maintenance of primitive peripheral blood-derived hematopoietic progenitor cells (HPC) cultured in serum-free medium supplemented with early acting cytokines

BMPs regulate the developmental program of hematopoiesis. We demonstrate an increased expression of the BMP receptors Ia and II on cultured CD34⁺ cells and examine the impact of BMP-2, -4 and -7 on postnatal HPC cultured with stem cell factor, flt3-ligand and interleukin-3 (SF3). The addition of BMP-2 at 5, 25 and 50 ng/m to serum-free medium with SF3 yielded a 1.4- to 1.2-fold increase of CD34⁺ cells after seven days, but no effect on CFC or LTC-IC was observed. BMP-4 at 25 ng/ml induced a 2.9-fold expansion of colony-forming cells (CFC) within 1 week followed by a decrease to pre-culture values on day 14. The number of long-term culture initiating cells (LTC-IC) decreased by the factor 40 from day 0 to day 14. BMP-7 at 5–50 ng/ml had not effect on the expansion of CD34⁺ cells and CFC, but improved at 5 ng/ml the survival of LTC-IC significantly as compared to SF3 alone. In summary, BMP-2, -4 and -7 have no effect on the proliferation of CD34⁺ cells and CFC cultured with serum-free medium and SF3. However, BMP-7 but not BMP-2 and BMP-4 prevents the loss of primitive hematopoietic progenitor cells cultured in SFM plus SF3.     

The generation of anti-tumoral cells using dentritic cells from the peripheral bloood of patients with malignant brain tumors

 Dendritic cells (DCs) can be the principal initiators of antigen-specific immune responses. We analyzed the in vitro-responses against brain tumor cells using DCs from the peripheral blood of patients with brain tumors. Peripheral blood mononuclear cells (PBMC) were obtained from 19 patients with malignant brain tumors: 12 metastatic brain tumors of lung adenocarcinoma, 7 high-grade astrocytomas. PBMC were cultured with 100 ng/ml of GM-CSF and 10 ng/ml of IL-4 for 5–7 days in order to produce mature DCs. The autologous tumor lysate (5 mg/ml, containing 1 × 10⁶ cells) was then added to the cultured DCs. Using the DCs generated by these treatments, we assessed the changes that occurred in their immune responses against brain tumor via ⁵¹Cr-release and lymphocyte proliferation assays. We found that the matured DCs displayed the typical surface phenotype of CD3⁺, CD45⁺, CD80⁺ and CD86⁺. After the pulsation treatment with tumor lysate, DCs were found to have strong cytotoxic T lymphocyte activity, showing 42.5 ± 12.7% killing of autologous tumor cells. We also found an enhancement of allogeneic T cell proliferation after pulsing the DC with tumor lysate. These data support the efficacy of DC-based immunotherapy for patients with malignant brain tumors. Received: 2 October 2000 / Accepted: 26 April 2001     

The frequency, growth kinetics, and osteogenic/adipogenic differentiation properties of canine bone marrow stromal cells

Bone marrow stromal cells (BMSCs) have gained considerable attention as a potential source for cell transplantation therapies for a variety of diseases due to their accessibility, proliferative capacity, and multilineage differentiation properties. Canine BMSCs have been shown to contribute to regeneration of osseous tissues, but knowledge about their biology is currently limited. In the present study, we investigated the frequency of adult canine BMSCs in bone marrow, morphological features, growth kinetics, and osteogenic as well as adipogenic differentiation properties in vitro. Our data suggest that adult canine bone marrow contains approximately one BMSC in every 2.38 × 10⁴ bone marrow mononucleated cells (0.0042 ± 0.0019%, n = 5). Primary BMSC cultures consisted of morphologically heterogeneous adherent cell populations from which spindle-shaped cells grew and became the predominant cell type. Growth kinetics patterns were dependent on the initial cell seeding densities, resulting in the highest fold increase at lower cell density. In the presence of osteogenic and adipogenic inducers, primary BMSCs underwent morphological and phenotypic changes characteristic of osteogenic and adipogenic differentiation, respectively. This study provides insights into basic characterization of adult canine BMSCs.     

The promoting effect of retinoic acid on proliferation of chicken primordial germ cells by increased expression of cadherin and catenins

Proliferation and cellular aggregation are both crucial features for survival and self-renewal of primordial germ cells (PGCs). Adhesive proteins play pivotal roles in cell–cell adhesion and signal exchanges under the influence of cytokines, growth factors and bioactive metabolites such as retinoic acid (RA). In this study, proliferation-promoting effect of RA on chicken PGCs was investigated by revealing changes in adhesive proteins E-cadherin and α/β catenins. PGCs were isolated from the genital ridge of 4-day-old chicken embryos and cultured on embryonic fibroblast feeder. RA (10⁻⁷–10⁻⁵ M) increased PGCs aggregation and mRNA expression of E-cadherin and α/β-catenins. Furthermore, E-cadherin and β-catenin protein expression levels were increased by RA treatment. However, RA-elicited effect was significantly attenuated by a PKC inhibitor H₇. In addition, the number and area of PGC colonies were increased by RA treatment at 10⁻⁷–10⁻⁵ M. Again, this increase was reduced by combined treatment of H₇. The proliferating effect of RA on PGCs was further confirmed by increased mRNA expression of cyclins, CCND1 and CCNE1, and cyclin-dependent kinases 6 and 2, which are critical for G1–S progression in cell cycle. Moreover, flow cytometry analysis confirmed that RA-treated PGC populations displayed a significant increase in the proportion of S and G2 phase cells. Likewise, this stimulating action was hindered by combined H₇ treatment. These results indicate that RA, as a bioactive metabolite of vitamin A, may promote PGC proliferation and increase intercellular aggregation of PGCs via E-cadherin and α/β-catenins expression through the PKC signaling pathway.     

In vitro propagated dendritic cells from patients with human-papilloma virus-associated preneoplastic lesions of the uterine cervix: use of Flt3 ligand

Dendritic cells (DC) are the most efficient antigen presenting cells. The clinical use of DC as vectors for antitumor and anti-infectious disease immunotherapy has been limited by their low level and accessibility in normal tissue. Substantial numbers of DC can be generated from peripheral blood cultured in the presence of interleukin-4 (IL-4) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). We showed in this study that substantial numbers of DC can be obtained from the peripheral blood of patients with (pre)neoplastic lesions of the uterine cervix. The procedure required relatively small blood samples (10 ml) and the presence of 100 U/ml IL-4 and 800 U/ml GM-CSF in the culture medium. There was no significant difference in the morphology, yield, phenotype and function of generated DC between patients with cervical (pre)neoplastic lesions and healthy individuals. When the hematopoietic factor Flt3 ligand (Flt3L, 40 ng/ml) was added, there was an average increase in the DC population of 26% compared to cultures with GM-CSF and IL-4 alone. Approximately 1.2 × 10⁶ cells with the characteristics of dendritic cells could be obtained when Flt3L was included in the medium. The addition of Flt3L did not modify the phenotypic profile of DC (HLA-DR⁺, CD1a⁺, CD4⁺, CD54⁺, CD80⁺, CD86⁺, CD40⁺, CD3⁻ and CD14⁻). In addition, Flt3L generated functional DC capable of stimulating the proliferation of alloreactive T cells. These results suggest that Flt3L, in association with GM-CSF and IL-4, provides an advantageous tool for the large-scale generation of DC and that an immunotherapy based on the use of DC generated in vitro is possible in patients with (pre)neoplastic lesions of the uterine cervix. Received: 8 January 1998 / Accepted: 30 April 1998     

Simultaneous measurement of soluble carcinoembryonic antigen and the tissue inhibitor of metalloproteinase TIMP1 serum levels for use as markers of pre-invasive to invasive colorectal cancer

Matrix metalloproteinases (MMP) are members of a multigene family of zinc-dependent enzymes involved in the degradation of extracellular matrix components. Cancer research suggest that MMP and tissue inhibitors of metalloproteinases (TIMP) may be involved in disease progression; these enzymes could therefore be used as markers in cancer prevention programmes and for clinical monitoring. To establish whether MMP and TIMP can be used effectively as markers we determined serum levels of MMP1 and TIMP1, and studied the relationships between these enzymes and the stage of disease. The potential diagnostic and prognostic value of serum level measurements of MMP1 and TIMP1 was evaluated by comparing them with serum levels of soluble carcinoembryonic antigens (sCEA) and p53 antibodies. Our overall results indicate that simultaneous measurements of serum sCEA and TIMP1 in patients with colorectal cancer could be used as prognostic and diagnostic markers for disease progression from the pre-invasive nodal phase to the invasive phase (stages I, II to III, IV). In addition, serum levels of TIMP1 could be used as a selective marker for metastatic disease (stage III to IV). In fact, the 95% confidence interval of the serum levels of sCEA at stage III (18.4 ≤ sCEA ≤ 68.6 ng/ml) and TIMP1 at stage IV (1620 ≤ TIMP1 ≤ 3906 ng/ml) identified statistically significant ranges of values (sCEA P = 0.02, TIMP1 P = 0.02), which may be useful in the monitoring of patients at these disease phases. More specifically, our data suggest that, when the serum level of sCEA is below 18.4 ng/ml and the level of TIMP1 below 1620 ng/ml, there is a 95% probability that the disease is in the pre-invasive nodal phase; when the serum level of sCEA falls between 18.4 ng/ml and 68.6 ng/ml and the level of TIMP1 is below 1620 ng/ml, there is a 95% probability that the disease is in the phase when lymph node infiltration occurs; when the level of sCEA is above 68.6 ng/ml and the level of TIMP1 is at least 1620 ng/ml, there is a 95% probability that the disease is in the metastatic phase. Received: 30 March 2000 / Accepted: 18 May 2000     

Plasma Levels of Zinc,Copper, and Ceruloplasmin in Patients after Undergoing Laparoscopic Adjustable Gastric Banding

Laparoscopic adjustable gastric banding (LAGB) causes significant weight loss in morbidly obese adults. However, its consequences on nutritional status still remain unclear. There are a few studies determining the nutritional status after LAGB and none have focused on the serum levels of zinc (Zn), copper (Cu), and ceruloplasmin (CP). We aimed to investigate the effects of LAGB surgery on plasma Zn, Cu, and CP levels. Thirty patients with LAGB with morbid obesity were included. Blood samples were collected preooperatively and in the postoperative third month to determine plasma Zn, Cu, and CP levels. The mean preoperative and postoperative body mass indexes (BMI) were 44.9 ± 7.4 kg/m² and 44.1 ± 6.5 kg/m², respectively. The mean weight loss was 12.9 ± 3.3 kg at the postoperative third month. The postoperative Zn (500 ± 130 ng/ml), Cu (280 ± 80 ng/ml), and CP (23.9 ± 8.8 mg/dl) values were statistically significantly lower than the preooperative Zn (740 ± 230 ng/ml), Cu (370 ± 80 ng/ml) and CP (33.3 ± 15.7 mg/dl) levels (p < 0.05). Decreases in the plasma levels of Zn, Cu, and CP were seen postoperatively following LAGB surgery. The nutritional status of LAGB-applied patients should be monitored and mineral supplementation may be considered.     

The Impact of High-dose Sodium Selenite Therapy on Bcl-2 Expression in Adult Non-Hodgkin’s Lymphoma Patients: Correlation with Response and Survival

The present study was undertaken to explore the effect of administration of high doses of sodium selenite on the expression of Bcl-2 in patients with non-Hodgkin’s lymphoma (NHL). Fifty patients with newly diagnosed NHL were randomly divided into two groups. Group A-I received standard chemotherapy whereas group A-II received adjuvant sodium selenite 0.2 mg kg⁻¹ day⁻¹ for 30 days in addition to chemotherapy. Enzyme-linked immunosorbent assay was used to assess Bcl-2 at the time of diagnosis and after therapy in the two groups. Sodium selenite administration resulted in significant decline of Bcl-2 level after therapy in group A-II (8.6 ± 6.9 ng/ml vs 3 6.9 ± 7.9 ng/ml, P < 0.05). Also, complete response reached 60% in group A-II compared to 40% in group A-I. Significant increase in CD4/CD8 ratio was noticed in group A-II compared to group A-I after therapy (1.45 ± 0.36 vs 1.10 ± 0.28 p 0.04). Overall survival time in months was significantly longer in complete remission patients in group A-II (21.87 ± 1.41) compared to group A-I (19.70 ± 1.95) (p = 0.01). It is concluded that sodium selenite administration at the dosage and duration chosen acts as a downregulator of Bcl-2 and improves clinical outcome.     

Dynamic Changes of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> Regulatory T Cells in NOD.H-2<Superscript>h4</Superscript> Mice with Iodine-Induced Autoimmune Thyroiditis

Iodine is an essential trace element for thyroid hormone synthesis and metabolism, either low or high intake may lead to thyroid disease, but the pathogenetic mechanisms by which iodine interacts with the thyroid autoimmune are poorly understood. We investigated the dynamic changes of CD4⁺CD25⁺ regulatory T cells in NOD.H-2^h4 mice with iodine-induced autoimmune thyroiditis (AIT), and explore potential immune mechanism of AIT induced by iodine. NOD.H-2^h4 mice were randomly divided into two groups, and received plain water or water containing 0.005% sodium iodide. Eight weeks after iodine provision, the incidences of thyroiditis, relative weights of thyroids, and serum thyroglobulin antibody titers in the iodine-supplied groups were significantly increased compared to the control groups (p < 0.05). The AIT mice had fewer CD4⁺CD25⁺Foxp3⁺ T cells and reduced Foxp3 mRNA expression in splenocytes compared with the controls (p < 0.01), and maintained relatively low levels during the development of thyroiditis. The changes described above aggravated gradually with the extension of iodine treatment. These data suggest that CD4⁺CD25⁺ regulatory T cells may be involved in the pathogenesis and development of AIT induced by iodine.     

Long-term treatment with low doses of interleukin-2 and interferon-α: immunological effects in advanced renal cell cancer

We aimed to determine the immunological effects of low doses of recombinant interleukin-2 (rIL-2) and recombinant interferon-α (rIFN-α) in patients bearing advanced renal cell carcinoma. Methods: Twenty-seven patients received therapeutic cycles consisting of subcutaneous rIL-2 for 5 days per week and intramuscular rIFN-α twice weekly, for 4 consecutive weeks. The cycle was repeated indefinitely at regular 4-month intervals, for all patients. rIL-2 (1 × 10⁶ IU/m²) was administered every 12 h on days 1 and 2 and once a day on days 3–5 of each week; rIFN-α (1.8 × 10⁶ IU/m²) was given on days 3 and 5. In the enrolled patients, total and differential white blood cell counts, phenotypic analysis of some lymphocyte subsets, and soluble IL-2 receptor (sIL-2R), were investigated before and after each of the first six cycles of therapy (about 24 months of follow-up). Results: The cycles of immunotherapy induced a significant increase of total lymphocytes (37%, P < 0.001), eosinophils (222%, P < 0.001), CD25+ cells (27%, P=0.004), sIL-2R (174%, P < 0.001) and natural killer (NK) cells (CD3-CD56+) (61%, P < 0.001); the subset that expresses CD56 with high density (CD56+ bright) expanded more (233%, P < 0.001) than the subset expressing the same marker with low density (CD56+ dimmer) (15%, P=0.043). Unlike the previous subsets, the treatment decreased significantly T-lymphocytes with NK cell marker (CD3+ CD56+) (28%, P=0.011). No significant differences of effectiveness were found among the subsequent treatment cycles, except for CD25+ cells and sIL-2R (P=0.036 and P=0.005, respectively): the increase induced by immunotherapy was maximum after the first cycle and decreased progressively thereafter. Conclusions: Long-term repeated cycles of low-dose immunotherapy induced repeated and significant expansion of one of the most important lymphocyte subsets for the non-MHC-restricted immune response to the tumour mass: CD3–CD56+ cells. Received: 8 November 2000 / Accepted: 11 January 2001     

Effect of Dietary Selenium and Cancer Cell Xenograft on Peripheral T and B Lymphocytes in Adult Nude Mice

Selenium (Se) is known to regulate tumorigenesis and immunity at the nutritional and supranutritional levels. Because the immune system provides critical defenses against cancer and the athymic, immune-deficient NU/J nude mice are known to gradually develop CD8⁺ and CD4⁺ T cells, we investigated whether B and T cell maturation could be modulated by dietary Se and by tumorigenesis in nude mice. Fifteen homozygous nude mice were fed a Se-deficient, Torula yeast basal diet alone (Se−) or supplemented with 0.15 (Se+) or 1.0 (Se++) mg Se/kg (as Na₂SeO₄) for 6 months, followed by a 7-week time course of PC-3 prostate cancer cell xenograft (2 × 10⁶ cells/site, 2 sites/mouse). Here, we show that peripheral B cell levels decreased in nude mice fed the Se −  or Se++ diet and the CD4⁺ T cell levels increased in mice fed the Se++ diet. During the PC-3 cell tumorigenesis, dietary Se status did not affect peripheral CD4⁺ or CD8⁺ T cells in nude mice whereas mice fed with the Se++ diet appeared to exhibit greater peripheral CD25⁺CD4⁺ T cells on day 9. Dietary Se status did not affect spleen weight in nude mice 7 weeks after the xenograft. Spleen weight was associated with frequency of peripheral CD4⁺, but not CD8⁺ T cells. Taken together, dietary Se at the nutritional and supranutritional levels regulates peripheral B and T cells in adult nude mice before and after xenograft with PC-3 prostate cancer cells.     

Angiotensin converting enzyme gene polymorphism is associated with severity of coronary artery disease in men with high total cholesterol levels

This study examines whether renin-angiotensin-aldosterone system gene polymorphisms: ACE (encoding for angiotensin converting enzyme) c.2306-117_404 I/D, AGTR1 (encoding for angiotensin II type-1 receptor) c.1080*86A>C and CYP11B2 (encoding for aldosterone synthase) c.-344C>T are associated with the extension of coronary atherosclerosis in a group of 647 patients who underwent elective coronary angiography. The extension of CAD was evaluated using the Gensini score. The polymorphisms were determined by PCR and RFLP assays. The associations between genotypes and the extent of coronary atherosclerosis were tested by the Kruskal-Wallis test, followed by pairwise comparisons using Wilcoxon test. The population has been divided into groups defined by: sex, smoking habit, past myocardial infarction, BMI (>, ≤ 25), age (>, ≤ 55), diabetes mellitus, level of total cholesterol (>, ≤ 200 mg/dl), LDL cholesterol (>, ≤ 130 mg/dl), HDL cholesterol (>, ≤ 40 mg/dl), triglycerides (>, ≤ 150 mg/dl). Significant associations between the ACE c.2306-117_404 I/D polymorphism and the Gensini score in men with high total cholesterol levels (P_{Kruskal-Wallis} = 0.008; P_adjusted = 0.009), high level of LDL cholesterol (P_{Kruskal-Wallis} = 0.016; P_adjusted = 0.028) and low level of HDL cholesterol (P_{Kruskal-Wallis} = 0.04; P_adjusted = 0.055) have been found. No association between the AGTR1 c.1080*86A>C and CYP11B2 c.-344C>T and the Gensini score has been found. These results suggest that men who carry ACE c.2306-117_404 DD genotype and have high total cholesterol, high LDL cholesterol and low HDL cholesterol levels may be predisposed to the development of more severe CAD.     

Intracellular cytokine profile of T cells from children with acute lymphoblastic leukemia

Purpose: During an ongoing immune response, cytokines produced by T helper types 1 (Th1) and 2 (Th2) together with T cytotoxic types 1 (Tc1) and 2 (Tc2) are critical to the effectiveness of that response. Dysregulated expansion of one or the other subset may contribute to the impaired function of the T-cell-mediated immune system in cancer patients. In the present study we have investigated whether such dysregulation might exist in children with acute lymphoblastic leukemia (ALL). Methods: We analyzed 61 blood samples from 45 children with B cell precursor ALL and 16 healthy children. Interleukin(IL)-2, IL-4, and interferon γ (IFNγ) production of their respective purified CD4⁺ and CD8⁺ T cells were assessed at the single-cell level by intracellular-cytokine-staining flow cytometry. Results: At the time of diagnosis, IL-2-producing cell populations in CD4⁺ and CD8⁺ T cells were reduced below the normal range in 31 of 44 (70.5%) and 23 of 38 (60.5%) cases respectively. Similarly, IFNγ-producing cell populations in CD4⁺ and CD8⁺ T cells decreased in 17 of 44 (38.6%) and 18 of 38 (47.4%) cases respectively. Conversely cell populations capable of IL-4 production in CD4⁺ and CD8⁺ T cell subsets were increased in 13 of 30 (43.3%) and 15 of 30 (50.0%) cases respectively. Therefore, the Th1-to-Th2 and Tc1-to-Tc2 ratios (1.6 ± 2.2 and 7.7 ± 6.7 respectively) were significantly lower in peripheral blood T cells of ALL patients (n = 30) than those (6.0 ± 2.9 and 20.1 ± 10.3 respectively) in 15 healthy controls (P < 0.0001). Although both CD45RA⁺/CD4⁺ and CD45RA⁺/CD8⁺ cells significantly increased in 43 ALL patients (P < 0.05), there existed no apparent correlation between CD45 isoform expression and cytokine (IL-2 and IFNγ) production. Interestingly, the ability to produce both IL-2 and IFNγ was recovered in 8 cases examined, after complete remission had been achieved. Conclusion: These observations suggest that, in both CD4⁺ and CD8⁺ T cells of ALL patients, there is a dysregulation in the functionality of Th1 (Tc1) and Th2 (Tc2) cells with a gross reduction of Th1 (Tc1) cell populations and an expansion in Th2 (Tc2). Received: 12 November 1999 / Accepted: 2 January 2000     

Optimization of protoplast yields from the red algae <Emphasis Type="Italic">Gracilaria dura</Emphasis> (C. Agardh) J. Agardh and G. <Emphasis Type="Italic">verrucosa</Emphasis> (Huds.) Papenfuss

This study reports on the optimization of protoplast yield from two important tropical agarophytes Gracilaria dura and Gracilaria verrucosa using different cell-wall-degrading enzymes obtained from commercial sources. The conditions for achieving the highest protoplast yield was investigated by optimizing key parameters such as enzyme combinations and their concentrations, duration of enzyme treatment, enzyme pH, mannitol concentration, and temperature. The significance of each key parameter was also further validated using the statistical central composite design. The enzyme composition with 4% cellulase Onozuka R-10, 2% macerozyme R-10, 0.5% pectolyase, and 100 U agarase, 0.4 M mannitol in seawater (30‰) adjusted to pH 7.5 produced the highest protoplast yields of 3.7 ± 0.7 × 10⁶ cells g⁻¹ fresh wt for G. dura and 1.2 ± 0.78 × 10⁶ cells g⁻¹ fresh wt for G. verrucosa when incubated at 25°C for 4–6 h duration. The young growing tips maximally released the protoplasts having a size of 7–15 μm in G. dura and 15–25 μm in G. verrucosa, mostly from epidermal and upper cortical regions. A few large-size protoplasts of 25–35 μm, presumably from cortical region, were also observed in G. verrucosa.     

Rice germ oil as multifunctional excipient in preparation of self-microemulsifying drug delivery system (SMEDDS) of tacrolimus

Surmounting the constraints of limited solubilization efficiency and prime requisite of antioxidant for conventional lipid formulations, the research work explores an edge over formulation utilizing potential applicability of rice germ oil (RGO) as a multifunctional excipient. Self-microemulsifying drug delivery system (SMEDDS) of tacrolimus (TAC) was formulated with RGO, an indigenous source of gamma-oryzanol. Being the same biological source, RGO and rice bran oil (RBO) were compared and it was found that RGO have more solubilization potential for TAC (2.2-fold) as well as higher antioxidant activity (8.06-fold) than the RBO. TAC-SMEDDS was prepared using RGO/Capmul PG8 (2:3) as an oil phase, Cremophore EL as a surfactant, and Transcutol P as a cosurfactant. The approximate particle size of TAC-SMEDDS was found to be 38 nm by dynamic light scattering and 12 nm by small angle neutron scattering. The in vitro dissolution studies showed complete and rapid drug release in 30 min compared to a plain drug (<5%) and marketed capsule (<50%). AUC and C _max were found to be 45.05 ± 15.64 ng h/ml and 3.91 ± 1.2 ng/ml for TAC-SMEDDS, 12.59 ± 5.54 ng h/ml and 0.48 ± 0.12 ng/ml for plain TAC, and 30.23 ± 10.34 ng h/ml and 2.31 ± 0.68 ng/ml for marketed formulation, respectively. The improved pharmacokinetic profile of TAC-SMEDDS is correlating to the dissolution results. Thus, gamma-oryzanol-enriched RGO acts as a potential multifunctional excipient for lipid formulations.