Inducible expression of dominant negative insulin-like growth factor I receptor in MCF-7 breast cancer cells

The insulin-like growth factor I receptor (IGF-IR) is expressed in many cell types and is critical for normal growth and development. In the healthy mammary gland, the role of IGF-IR is not fully elucidated. However, IGF-IR, which is primarily expressed in the mammary epithelial cells, is known to play an obligatory role in cellular transformation, facilitating the progression to breast cancer. We have utilized the tetracycline regulatory (tet-on) system to generate an in vitro model system to allow us to further investigate IGF-I/IGF-IR function in mammary epithelial cells. A plasmid construct containing a mutant IGF-I receptor (IGF-IR-DN) fused to the tetracycline operator (tetOPh_CMV-IGF-IR-DN) was stably transfected into MCF-7 human breast cancer cells. The conditional regulation of the IGF-IR-DN gene expression was studied in four independent clonal lines. The translated IGF-IR-DN protein was detected only in the stably transfected doxycycline-induced cells, and its expression was up-regulated (three- to sixfold) following induction. IGF-I stimulated cell proliferation diminished (twofold) in doxycycline-induced cells compared to uninduced cells, demonstrating that the transgene construct was functional and ruling out any pleiotropic effect that may be attributed to doxycycline. Interestingly, autophosphorylation of the IGF-IR and phosphorylation of the downstream substrate, insulin receptor substrate-1 (IRS-1), was not inhibited in doxycycline/IGF-I treated cells, suggesting the possibility that activation of downstream substrates other than the IRS-1 may be critical for optimal cell proliferation. This novel in vitro model should allow us to more directly examine the role of IGF-I/IGF-IR signaling and function in mammary epithelial cells.     

Chondrocalcinosis secondary to hypomagnesemia in Gitelman's syndrome

Chondrocalcinosis can be associated with hyperparathyroidism, hemochromatosis, hypophosphatasia, and hypomagnesemia. Gitelman syndrome (GS), an inherited disorder due to loss of function mutations of the gene encoding the distal convoluted tubule Na-Cl cotransporter (NCCT), is characterized by hypokalemia metabolic alkalosis, hypomagnesemia, and hypocalciuria. A 53-year-old man, with history of recurrent joint effusions and pains affecting knees and wrists, had transient episodes of muscle pain, weakness, cramping, and fatigue over a one-year period. Laboratory tests showed hypokalemia, metabolic alkalosis, hypocalciuria, and hypomagnesemia related to genetically proven GS. Radiographs of affected joints revealed calcium pyrophosphate dihydrate deposition. This observation points out the necessity to look for Mg depletion (and especially GS) in the biological investigation of chondrocalcinosis. Additionally, the association between GS (NCCT inactivation) and high bone mineral density provides a new insight into the possible role of thiazides in osteoporosis management.     

Red cell membrane stiffness in iron deficiency

The purpose of this study was to characterize red blood cell (RBC) deformability by iron deficiency. We measured RBC deformability to ektacytometry, a laser diffraction method for determining the elongation of suspended red cells subjected to shear stress. Isotonic deformability of RBC from iron-deficient human subjects was consistently and significantly lower than that of normal controls. In groups of rats with severe and moderate dietary iron deficiency, RBC deformability was also reduced in proportion to the severity of iron deficiency. At any given shear stress value, deformability of resealed RBC ghosts from both iron-deficient humans and rats was lower than that of control ghosts. However, increase of applied shear stress resulted in progressive increase in ghost deformation, indicating that ghost deformability was primarily limited by membrane stiffness rather than by reduced surface area-to-volume ratio. This was consistent with the finding that iron-deficient cells had a normal membrane surface area. In addition, the reduced mean corpuscular hemoglobin concentration (MCHC) and buoyant density of the iron-deficient rat cells indicated that a high hemoglobin concentration was not responsible for impaired whole cell deformability. Biochemical studies of rat RBC showed increased membrane lipid and protein crosslinking and reduced intracellular cation content, findings that are consistent with in vivo peroxidative damage. RBC from iron-deficient rats incubated in vitro with hydrogen peroxide showed increased generation of malonyldialdehyde, an end-product of lipid peroxidation, compared to control RBC. Taken together, these findings suggest that peroxidation could contribute in part to increased membrane stiffness in iron- deficient RBC. This reduced membrane deformability may in turn contribute to impaired red cell survival in iron deficiency.     

Characterizing the PK/PD relationship for inhibition of capsaicin-induced dermal vasodilatation by MK-3207, an oral calcitonin gene related peptide receptor antagonist

Aims

Calcitonin gene related peptide (CGRP) receptor antagonists are effective acute migraine treatments. A capsaicin-induced dermal vasodilatation (CIDV) model has been developed to provide target-engagement information in healthy volunteers. In the model, CGRP release is provoked after dermal capsaicin application, by activating transient receptor potential vanilloid-type-1 (TRPV1) receptors at peripheral sensory nerves. Laser Doppler imaging is used to quantify CIDV and subsequent inhibition by CGRP receptor antagonists. We sought to evaluate a CGRP receptor antagonist, MK-3207, in the biomarker model and to assess the predictability of the CIDV response to migraine clinical efficacy.

Methods

An integrated population pharmacokinetic/pharmacodynamic (PK/PD) model was developed to describe the exposure−response relationship for CIDV inhibition by CGRP and TRPV1 receptor antagonists. MK-3207 dose−response predictions were made based on estimated potency from the PK/PD model and mean plasma concentrations observed at the doses investigated.

Results

The results suggested that a 20 mg dose of MK-3207 (EC₅₀ of 1.59 nm) would be required to attain the peripheral CIDV response at a target level that was shown previously to correlate with 2 h clinical efficacy based on phase 3 telcagepant clinical data, and that a plateau of the dose−response would be reached around 40–100 mg. These predictions provided a quantitative rationale for dose selection in a phase 2 clinical trial of MK-3207 and helped with interpretation of the efficacy results from the trial.

Conclusions

The integrated CIDV PK/PD model provides a useful platform for characterization of PK/PD relationships and predictions of dose−response relationships to aid in future development of CGRP and TRPV1 receptor antagonists.     

Using a focus group to design a diabetes education program for an African American population

PURPOSE: The purpose of this project was to determine what factors need to be considered in planning a diabetes education program to better meet the needs of African Americans with diabetes in a community served by a suburban community hospital. METHODS: Two focus group sessions were conducted. The sessions were recorded, transcribed, and analyzed by members of the research team. RESULTS: Four themes emerged that had bearing on program development: (1) a sense of personal powerlessness, (2) fear related to complications, (3) recognition of knowledge deficits accompanied by an inability to link behavior to outcomes, and (4) a clear vision of what kind of educational setting would interest and benefit the group. CONCLUSIONS: There were significant differences between what was being offered for diabetes education at the facility and what was desired according to the focus group, including factors of cost and leadership. Recommendations for future program planning are given.     

Comparison of the native prothrombin antigen and the prothrombin time for monitoring oral anticoagulant therapy

We have measured the fully carboxylated (native) prothrombin antigen and the undercarboxylated (abnormal) prothrombin antigen in patients treated with sodium warfarin using specific immunoassays to evaluate a new approach for monitoring oral anticoagulant therapy. Plasma and serum samples (391) were assayed for the prothrombin time, native prothrombin antigen, and abnormal prothrombin antigen. The results were correlated with the presence of bleeding or thromboembolic complications at the time of phlebotomy. The native prothrombin antigen correlated with the occurrence of complications in 95% of samples. Of 13 samples from patients with bleeding complications, 13/13 (100%) had a native prothrombin of 12 micrograms/mL or lower. Of seven samples from patients with thromboembolic complications, 6/7 (86%) had a native prothrombin of 24 micrograms/mL or greater. By comparison, a prothrombin time index of 1.5 to 2.5, 1.5 to 2.2, 1.5 to 2.0, or 1.3 to 1.8 identified 6/20 (30%), 9/20 (45%), 11/20 (55%), or 12/20 (60%) patients at risk, respectively. Although the prothrombin time index did correlate with the presence of bleeding complications, the native prothrombin antigen correlated closely with the presence of bleeding and thromboembolic complications. According to these results, the native prothrombin antigen, maintained in a range of 12 to 24 micrograms/mL by regular adjustment of the warfarin dosage, may be associated with a reduced risk of complications due to excessive or insufficient warfarin therapy. On the basis of these preliminary data, we recommend that the native prothrombin antigen be considered to monitor warfarin therapy.     

Generational differences in the age at diagnosis with Ibd: genetic anticipation, bias, or temporal effects

OBJECTIVE: Previous cross-sectional research has demonstrated generational differences in age at diagnosis (AAD) in inflammatory bowel disease (IBD). This observation has at times been ascribed to genetic anticipation, but could also be due to biases related to case ascertainment or follow-up or to temporal changes in IBD epidemiology. We aimed to explore this issue using a population-based database. METHODS: In 1995 we used the comprehensive administrative databases in the province of Manitoba, Canada to establish a population-based IBD Research Registry that includes clinical and demographic information for persons. We contacted those subjects within our Research Registry who reported having any family members with IBD and their family members for verification of diagnosis and AAD. Differences in AAD between familial pairs were calculated. In addition, to assess whether duration of follow-up accounted for generational differences in AAD, we computed the mean AAD for subjects with and without family histories of IBD based on age at the time of interview (i.e., < 45 and > or = 45 yr of age). RESULTS: Of the 2445 persons with IBD in the Research Registry, 548 reported positive family histories, and 315 of these (58%) were reached by telephone. There were 169 Crohn's disease and 146 ulcerative colitis subjects with positive family histories. The mean AADs for the parents, aunts/uncles, and grandparents were significantly greater than the mean AADs for the children, nieces/nephews, and grandchildren, respectively. There was a doubling of the mean AAD when comparing the grandparent/grandchild cases with the parent/child or aunt/uncle-niece/nephew cases. No statistically significant difference in anticipation was observed, whether or not the older generation was male or female or had Crohn's disease or ulcerative colitis. The AAD was substantially greater for those interviewed at > or = 45 yr of age for subjects with and without family histories. However, there was no substantial difference in mean AAD between familial and nonfamilial subjects. CONCLUSION: The present study has demonstrated that there is a tendency for children to be younger than their parents at the time of diagnosis of familial IBD, and that this difference in mean AAD is almost doubled for grandparent/grandchild pairs. However, we conclude that these differences are most likely due to a bias based on length of follow-up or recent multigenerational temporal changes in the risk of IBD, or both.