Fenugreek improves diet-induced metabolic disorders in rats.

Trigonella foenum-graecum L. (fenugreek) has been described earlier and its use in ancient medicinal practice is well known. The hypoglycemic effects of fenugreek have been studied in many animal models and diabetic patients. The purpose of this study was to examine the preventive efficiency of dietary fenugreek on diet-induced metabolic diseases in rats. The diets used in this study were a standard diet, a high-fat high-sucrose (HFS) diet, and a HFS diet containing 0.5 g/kg b. w./day fenugreek based on the modified version of the AIN-93G purified diet, for 12 weeks, respectively. The rats fed the HFS diet containing fenugreek showed significantly lower fasting insulin levels and HOMA-IR than the rats fed the HFS diet. Therefore, fenugreek improved insulin sensitivity in rats. The triglyceride and total cholesterol levels in the plasma were significantly lower in the fenugreek-administered group. Moreover, distinct reductions of triglyceride, total cholesterol, free fatty acid, and phospholipid levels in the liver were found in the rats fed the HFS diet containing fenugreek. These results suggest that fenugreek enhanced insulin sensitivity at least partly by improving lipid metabolism disorders in the plasma and the liver in the rats induced by the HFS diet.

Paraneoplastic syndrome of hypercalcemia and leukocytosis caused by squamous carcinoma cells (T3M-1) producing parathyroid hormone-related protein, interleukin 1 alpha, and granulocyte colony-stimulating factor.

Previously we reported that a clonal squamous cell carcinoma cell line (T3M-1) derived from a lower jaw cancer of a patient with marked leukocytosis and hypercalcemia produced factors containing a potent bone-resorbing activity (BRA) (Mr 15,000-20,000) and a colony-stimulating activity. To elucidate the pathogenesis of this humoral hypercalcemia, BRA and colony-stimulating activity in both the conditioned medium and cells were characterized. The conditioned medium, when eluted at neutral pH, contained colony-stimulating activity and thymocyte proliferation-stimulating activity, the latter of which comigrated with BRA. Upon elution with acetic acid (pH 2.0), the conditioned medium contained no interleukin 1-like activity but potent parathyroid hormone-like activity, which comigrated with BRA. Northern blot hydridization analysis revealed that T3M-1 cells produced constitutively mRNA for parathyroid hormone-related protein and granulocyte colony-stimulating factor. Furthermore, primer extension analysis revealed that the cells also produced mRNA for interleukin 1 alpha (IL-1 alpha). Since parathyroid hormone-related protein and IL-1 alpha (osteoclast-activating factor) synergistically increase the concentration of serum calcium, and since IL-1 alpha (hemopoietin 1) potentiates granulocyte colony-stimulating factor-induced granulocytopoiesis, we speculate that parathyroid hormone-related protein, granulocyte colony-stimulating factor, and IL-1 alpha are synergistically involved in a paraneoplastic syndrome of hypercalcemia and leukocytosis, at least in some patients with solid tumors.

Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.

In vivo cancer gene therapy approaches for squamous cell carcinoma of the head and neck (SCCHN) based on adenoviral vector-mediated gene delivery have been limited by the suboptimal efficacy of gene transfer to tumor cells. We hypothesized that this issue was due to deficiency of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on the tumor targets. Studies of CAR levels on SCCHN cell lines confirmed that their relative refractoriness to the adenoviral vector was based on this deficiency. To circumvent this deficiency, we applied an adenoviral vector targeted to a tumor cell marker characteristic of SCCHN. In this regard, integrins of the alpha2beta1 and alpha3beta1 class are frequently overexpressed in SCCHN. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we applied an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. Studies confirmed that the CAR-independent gene delivery achieved via this strategy allowed enhanced gene transfer efficiencies to SCCHN tumor cells. Importantly, this strategy could achieve preferential augmentation of gene transfer in tumor cells compared with normal cells. The ability to achieve enhanced and specific gene transfer to tumor cells via adenoviral vectors has important implications for gene therapy strategies for SCCHN and for other neoplasms in general.

Urinary excretion of parathyroid hormone-related protein fragments in patients with humoral hypercalcemia of malignancy and hypercalcemic tumor-bearing nude mice.

To investigate whether parathyroid hormone-related protein (PTHrP), a hypercalcemia-inducing factor responsible for malignancy-associated hypercalcemia (MAH), is excreted into urine of these patients, radioimmunoassay was established using antiserum specific for the C-terminal region of PTHrP-(127-141). Immunoreactive PTHrP (iPTHrP) was detected in the urine of all patients with MAH (n = 6) in whom nephrogenous cyclic AMP excretion was elevated. However, iPTHrP was not detected in the urine of normal subjects (n = 25) or hypercalcemic patients with primary hyperparathyroidism (n = 8). In normocalcemic patients with malignant disorders iPTHrP was not detected in the urine in most cases (24 of 25 patients) but was detectable in 1 of 25 patients. iPTHrP was also detected in the urine of hypercalcemic nude mice transplanted with PTHrP-producing tumors, but not in the urine of control and normocalcemic nude mice transplanted with PTHrP-nonproducing tumor. Furthermore, size-exclusion high-performance liquid chromatography revealed that the molecular weight of iPTHrP is about 2000-6000 daltons in the urine of patients as well as tumor-bearing nude mice. These data indicate that the fragments of the C-terminal region of PTHrP are excreted into the urine of patients with MAH and in a few normocalcemic patients with malignancies, suggesting that the measurement of iPTHrP in the urine is potentially useful in the differential diagnosis of hypercalcemia, particularly in differentiating humoral hypercalcemia of malignancy and primary hyperparathyroidism.

Interleukin-4 inhibits spontaneous and parathyroid hormone-related protein-stimulated osteoclast formation in mice.

We examined the in vivo effects of recombinant murine IL-4 (rmIL-4) on spontaneous and stimulated mouse osteoclast formation. EC-GI cells, which produce PThrP and IL-1 alpha, were explanted in nude mice. These EC-GI cell-bearing nude mice developed hypercalcemia (4.90 +/- 0.68 mM), and the calcium levels were decreased to near normal (3.48 +/- 0.73 mM, p < 0.05) at day 3 by continuous infusion of rmIL-4 at a dose of 7 micrograms/day. When infused with 0.6 nmol/day of PTHrP(1-34) in ICR mice, rmIL-4 at a dose of 1 or 5 micrograms/day for 3 days caused a marked inhibitory effect on hypercalcemia induced by PTHrP(1-34) (3.73 +/- 0.56-2.54 +/- 0.14 mM, p < 0.01). However, rmIL-4 alone did not change the serum calcium in mice. Histomorphometric analysis revealed that rmIL-4 inhibits both spontaneous and PTHrP(1-34)-stimulated osteoclast formation in mice, with a decrease in osteoclastic surface and in the number of osteoclasts per mm bone surface, respectively. We conclude that IL-4 inhibits spontaneous and stimulated bone resorption resulting from inhibition of osteoclast formation and modulates the development of humoral hypercalcemia of malignancy.

Effect of interleukin-1 (IL-1) on thyroid hormone metabolism in mice: stimulation by IL-1 of iodothyronine 5'-deiodinating activity (type I) in the liver.

To elucidate the mechanism by which the low T3 and low T4 syndrome occurs in patients with infection, recombinant human interleukin-1 (IL-1) was administered to mice, and their thyroid hormone metabolism was studied. Continuous sc infusion of IL-1 alpha or IL-1 beta at a dose of 0.015-1 microgram/day for 3 days decreased food intake and serum T4, T3, and rT3 concentrations in a dose-dependent manner. In pair-fed control (PFC) mice, serum T4 and T3 also decreased, but rT3 was reciprocally increased. The T3/T4 ratio was greater in IL-1-treated mice than in PFC mice. Although food intake was decreased by 65% in IL-1-treated mice (1 microgram/day) compared with that in fed control mice, type I iodothyronine 5'-deiodinating activity in liver was significantly increased compared with that in fed control mice. Furthermore, the T3 and T4 responses to TSH were greatly diminished in IL-1-treated mice. These findings suggest that IL-1 directly inhibited the effect of TSH on the thyroid gland and decreased the serum concentrations of T4 and T3, and that an increase in type I iodothyronine 5'-deiodinating activity in livers of IL-1-treated mice may account for the greater T3/T4 ratio and lower serum rT3 concentration than those in PFC mice. Since tumor necrosis factor-alpha has a similar effect, we speculate that both cytokines may be synergistically involved in the altered thyroid hormone metabolism in mice (decreased serum T4, T3, and rT3 concentrations) and hypercatabolism in a febrile state.

Parathyroid hormone-related protein and interleukin-1 alpha synergistically stimulate bone resorption in vitro and increase the serum calcium concentration in mice in vivo.

To elucidate the mechanism of humoral hypercalcemia elicited by human esophageal carcinoma cells (EC-GI), which constitutively produced interleukin-1 alpha (IL-1 alpha) and PTH-like factor, the effects of IL-1 alpha and PTH-related protein (PTH-rP) on bone resorption in vitro and on serum calcium concentrations in vivo were investigated. Nude mice transplanted with EC-GI cells invariably developed hypercalcemia, although their urinary cAMP excretion remained within the normal range. IL-1 alpha or PTH-rP-(1-34) stimulated 45Ca release from prelabeled fetal mouse forearm bones in a concentration-dependent manner, and when combined, IL-1 alpha and PTH-rP-(1-34) synergistically stimulated bone resorption in vitro. Injection of PTH-rP-(1-34) into mice three times a day for 2 days increased the serum calcium concentration in a dose-dependent manner. Continuous infusion of IL-1 alpha occasionally increased the serum calcium concentration. Simultaneous administration of IL-1 alpha at rates of 1-2.7 micrograms/day and PTH-rP-(1-34) at doses of 15-30 micrograms/day synergistically increased the serum calcium concentration in vivo. These findings suggest that PTH-rP and IL-1 alpha produced by the tumor cells were synergistically responsible for the humoral hypercalcemia observed in both the original patient and the tumor-bearing nude mice, and that at least two bone-resorbing factors [PTH-rP and another nonadenylate cyclase-stimulating bone-resorbing factor(s)] are active in patients with malignancy-associated hypercalcemia, in whom nephrogenous cAMP excretion is neither increased nor decreased.

Production of interleukin-1 alpha and a parathyroid hormone-like factor by a squamous cell carcinoma of the esophagus (EC-GI) derived from a patient with hypercalcemia.

To determine the pathogenesis of humoral hypercalcemia associated with esophageal carcinoma in a 65-yr-old patient, clonal cell lines (EC-GI) were established from the tumor. The EC-GI cells produced bone-resorbing activity which eluted from a Sephadex G-75 column in a broad peak, with an apparent mol wt of 10,000-50,000. In addition to PTH-like factor(s), the EC-GI cells produced a factor with thymocyte proliferation-stimulating activity which had an apparent mol wt of 15,000-20,000. This interleukin-1 (IL-1)-like factor(s) with acidic pI (4.8 and 5.2) exactly coeluted with the bone-resorbing activity upon DEAE-Sepharose ion exchange chromatography. Both bone-resorbing and thymocyte proliferation-stimulating activities were completely inhibited by anti-IL-1 alpha antiserum, but not by anti-IL-1 beta antiserum. Northern blot hybridization studies revealed that EC-GI cells produced exclusively mRNA for IL-1 alpha. Furthermore, fractions containing IL-1-like activity and PTH-like activity synergistically stimulated bone resorption in vitro, and transplantation of EC-GI cells into nude mice caused hypercalcemia in vivo. These findings suggest that IL-1 alpha and PTH-like factor produced by this squamous cell carcinoma synergistically stimulate bone resorption and are related to humoral hypercalcemia in tumor-bearing nude mice and in the patient.

Falsely elevated serum parathyroid hormone levels due to immunoglobulin G in a patient with idiopathic hypoparathyroidism.

A 73-yr-old patient with idiopathic hypoparathyroidism was admitted to our hospital in May 1981. The immunoreactive PTH (iPTH) level determined by RIA using antiserum specific for the C-terminal region of PTH-(65-69) was in the upper normal range (0.6 ng/mL) and over the next 7 yr increased gradually to 6 ng/mL. Since iPTH levels determined using other commercial RIA kits remained constantly decreased or in the undetectable range, we studied the mechanism of false elevation of iPTH in this patient. The patient's serum contained no binding protein to the tracer ([125I]) [Tyr45] human PTH-(46-84)), nor was any heterophilic antibody to the first [guinea pig immunoglobulin G (IgG)] or the second antibody (goat IgG) detected. Consistent with these findings, the dilution curve of the serum was parallel with that of standard bovine PTH-(1-84). Gel filtration analysis revealed that the iPTH-like substance was eluted in the void volume (apparent mol wt, greater than 70,000). Almost all of the iPTH-like substance was adsorbod by a protein-A-Sepharose column. When the IgG fraction purified by protein-A-Sepharose affinity chromatography was applied to an antihuman IgG lambda-Sepharose column, 72% of the iPTH-like substance was detected in the IgG lambda. These results suggest that the falsely elevated iPTH in the patient's serum was due to IgGs (mainly IgG lambda), which were cross-reactive with the antiserum highly specific for the C-terminal region of human PTH-(65-69).

Tetracycline-induced expression of an anti-c-Myb single-chain antibody and its inhibitory effect on proliferation of the human leukemia cell line K562.

Ablation of c-Myb function might be an effective approach for the therapy of chronic myelogenous leukemia or other c-myb-dependent malignancies. To this end, we have previously used an intracellular anti-c-Myb single-chain antibody (sFv) to achieve the functional knockout of the c-Myb oncoprotein. In this study, we have employed a tetracycline-inducible system to control the expression of the sFv. A nuclear-localizing form of an anti-c-Myb sFv was cloned into a tet-regulated plasmid vector. Using a transient expression system in COS-1 cells, we observed that doxycycline (Dox) induced expression of the sFv in a dose-dependent manner, and that the sFv was localized mainly in the nucleus. The Dox-induced anti-c-Myb sFv also inhibited the transactivating activity of c-Myb in a dose-dependent manner. We subsequently confirmed the Dox-induced expression of the sFv in the leukemia cell line K562. Proliferation of the target leukemia cells was also inhibited. These results suggest that the anti-c-Myb sFv may represent a viable method for gene therapy of c-myb-dependent hematopoietic malignancies.