Mei X. Wu

Significant Differential Effects of Alendronate, Estrogen, or Combination Therapy on the Rate of Bone Loss after Discontinuation of Treatment of Postmenopausal Osteoporosis: A Randomized, Double-Blind, Placebo-Controlled Trial

Context Alendronate and conjugated estrogen therapy both increase bone mineral density in postmenopausal women, but is the rate of bone loss greater when alendronate or estrogen therapy is discontinued? Contribution The discontinuation phase of this double-blind, placebo-controlled trial showed loss of spine and trochanter bone mass in postmenopausal women 1 year after withdrawal of estrogen and no such loss after withdrawal of either alendronate or combination therapy with alendronate and estrogen therapy. Cautions The study was not large or long enough to show whether discontinuation of estrogen therapy is associated with more fractures than discontinuation of either alendronate or combination therapy. The Editors Several antiresorptive agents have been shown to increase bone mass and reduce osteoporotic fractures (1-3). Because greater improvements in bone mass in women using therapy are associated with greater reductions in fracture (4, 5), investigators have begun to examine combinations of antiresorptive therapies to achieve more substantial gains in bone mass. Lindsay and colleagues demonstrated that addition of alendronate to hormone replacement therapy in postmenopausal women resulted in greater increases in bone mass than did maintenance of estrogen therapy alone (6). We previously showed that administration of alendronate and estrogen for 2 years in postmenopausal women with low bone mass resulted in statistically significantly greater increases in bone mass at the lumbar spine and femoral neck than those seen in women taking either agent alone (7). Furthermore, combination therapy was safe and resulted in normal findings on histologic examination of bone. In clinical practice, a key concern is the potential for accelerated bone loss when antiresorptive therapy is discontinued. Approximately one third of women discontinue hormone replacement therapy within 1 year of initiation (8). Older studies have demonstrated significant losses in bone mass after discontinuation of hormone replacement therapy (9-11). In contrast, when therapy with oral alendronate, 10 mg/d, is discontinued after osteoporosis treatment, bone mass at the hip and spine are maintained for 1 year (12). However, no head-to-head comparison of hormone replacement therapy and alendronate or the combination of antiresorptive therapy after discontinuation has been done. In addition, future losses in bone mass when patients discontinue therapy must be considered in management of osteoporosis in postmenopausal women. We therefore sought to examine the rate of bone loss after discontinuation of 2 years of alendronate therapy, hormone replacement therapy, or combination therapy. A subset of participants continued to take combination therapy for a third year to determine whether prolonged therapy remained beneficial. Methods Study Participants Four hundred twenty-five postmenopausal women 42 to 82 years of age who had low bone mass were enrolled in a 2-year randomized, double-blind, placebo-controlled clinical trial conducted at 18 centers in the United States (7). Participants were recruited from clinics, private practices, newspaper advertisements, and targeted mailings. All participants who completed the initial study were asked to enroll in the 1-year extension. Participants were told that if they were taking active treatment, they might be randomly allocated to receive placebo or treatment for the third year and that if they were taking placebo, they would continue to do so. Entry criteria for the initial study are described elsewhere (7). All women had had hysterectomy and had a bone mineral density at the lumbar spine that was less than or equal to a T score of 2.0 SDs below the peak bone mass in young adults. Data on presence or absence of ovaries were not collected. Exclusion criteria were metabolic bone disease, a low serum 25-hydroxyvitamin D level, use of medications known to affect bone turnover, renal insufficiency, severe cardiac disease, and recent major upper gastrointestinal disease. The institutional review board at each clinical site approved the extension protocol. After signing the extension consent form and undergoing baseline evaluation for the extension, participants were allocated to blinded treatment on the basis of their original treatment in the first 2 years of the study. The randomization process was centrally determined by a statistician; as in the initial study, treatment allocation was concealed. Design As described for the initial study at each center, patients were randomly allocated to one of four treatment groups: placebo (n = 50); alendronate, 10 mg/d (n = 92); conjugated estrogen, 0.625 mg/d (n = 143); or alendronate, 10 mg/d, plus conjugated estrogen, 0.625 mg/d (n = 140) (Figure 1). The conjugated estrogen used was Premarin (Wyeth-Ayerst, Philadelphia, Pennsylvania). All women received calcium carbonate to provide 500 mg of elemental calcium daily. Figure 1. Design of original 2-year study and reallocation to extension phase for year 3. At the end of the second year, 244 of the 425 women (57%) continued in a 1-year extension of the study (Figure 1). Of these women, 28 who previously received placebo continued to do so. Women who were taking combination therapy were reallocated to continue taking combination therapy (n = 44) or switch to placebo (n = 41). In addition, 50 participants taking alendronate alone and 81 participants taking conjugated estrogen alone for the first 2 years were assigned to placebo for the third year. All patients and investigators remained blinded to medication allocation. Patients continued to receive calcium supplementation during the third year. Outcome Measures Women were examined at month 24 (baseline of the 1-year extension), month 30, and month 36. Bone mineral density of the lumbar spine, hip (femoral neck, trochanter, total hip), and total body were assessed by using dual-energy x-ray absorptiometry with QDR-1000W, QDR-1500, or QDR-2000 series bone densitometers (Hologic, Inc., Bedford, Massachusetts). A standard phantom was used for cross-calibration at all sites. Serum and urine samples were also obtained at months 24, 30, and 36 for assessment of biochemical markers of bone turnover, namely bone-specific alkaline phosphatase and urinary N-telopeptide cross-links of collagen type I, corrected for creatinine. Statistical Analysis We used SAS software, version 6.12, TSLevel 0060, PROCedureGLM (SAS Institute, Inc., Cary, North Carolina) to analyze the data. The primary efficacy end point was the mean difference between groups in the percentage change in bone mineral density at the lumbar spine from month 24 to month 36. Secondary efficacy end points were the mean percentage changes in bone mineral density of the hip and total body and biochemical markers of bone turnover. Overall percentage changes from month 0 to 36 in spine, hip, and total-body bone mineral density were also analyzed. The prespecified analysis was based on an intention-to-treat approach. At study design, we prespecified that all patients who had a baseline measurement and at least one measurement during treatment would be included in the analysis according to the group to which they were randomly allocated. The missing data were approximated by carrying forward the last available value on treatment forward to the missing time point. No data from the original 2-year study were carried forward to the extension period for any assessment of change. Women who violated the protocol were excluded from analysis of biochemical markers, as previously reported (7). Between-group comparisons of bone mineral density and biochemical measures were made by using analysis of variance techniques, with treatment, center, and treatment-by-center as factors. The assumption of homoscedasticity for the analysis of variance model was assessed by using the Levene test, and the normality assumption was assessed by using the ShapiroWilk test (13). If the assumptions were violated, a nonparametric method was used to corroborate the parametric results. The Fisher exact test was used to compare treatment groups for the proportion of participants who exceeded predefined limits of change in laboratory safety variables (13). Power calculations based on estimated sample sizes of 56 and 84 participants in the alendronate/placebo and estrogen/placebo treatment groups, respectively, yielded an estimate of 92% power to detect a 1.5% difference between mean percentage changes from month 24 to month 36 in bone mineral density at the lumbar spine ( = 0.05, two-tailed test). As requested by the journal editors, data on bone mineral density were also analyzed by using a mixed-model analysis, and results of this analysis are presented. An appropriate curvilinear function was fitted to the actual data, and the function was estimated by using all data available across time points for each participant. A model that regressed bone mineral density versus log (month + 1) provided the appropriate fit for the 3-year data and was used to analyze these data. The variable log (month + 1) was used because log (month) is undefined when month is 0, and log (month + 1) yields the value 0 at baseline. The fitted values from the model were used to obtain the percentage change during the period of interest. Data on bone mineral density from the mixed-model analyses are presented unless otherwise specified. Role of the Funding Source Data were collected by investigators at each study site with the support of Merck Research Laboratories, Rahway, New Jersey. Analyses were performed by statisticians at Merck & Co., Inc. Data were interpreted by the authors, who submitted the manuscript for publication. Results Patient Characteristics and Retention Baseline randomization characteristics did not differ between participants who entered the extension phase and those who did not. Baseline demographic characteristics of the 244 women who entered the extension phase were s

Photodynamic therapy plus low-dose cyclophosphamide generates antitumor immunity in a mouse model

Photodynamic therapy (PDT) is a modality for the treatment of cancer involving excitation of nontoxic photosensitizers with harmless visible light-producing cytotoxic reactive oxygen species. PDT causes apoptosis and necrosis of tumor cells, destruction of the tumor blood supply, and activation of the immune system. The objective of this study was to compare in an animal model of metastatic cancer PDT alone and PDT combined with low-dose cyclophosphamide (CY) a treatment that has been proposed to deplete regulatory T cells (T-regs) and increase the immune response to some tumors. We used J774 tumors (a highly metastatic reticulum cell sarcoma line) and PDT with benzoporphyrin derivative monoacid ring A, verteporfin for injection (BPD; 1-mg/kg injected i.v. followed after 15 min by 150 J/cm2 of 690-nm light). CY (50 or 150 mg/kg i.p.) was injected 48 h before light delivery. PDT alone led to tumor regressions and a survival advantage but no permanent cures were obtained. BPD–PDT in combination with low-dose CY (but not high-dose CY) led to 70% permanent cures. Low-dose CY alone gave no permanent cures but did provide a survival advantage and was shown to reduce CD4+FoxP3+ T-regs in lymph nodes, whereas high-dose CY reduced other lymphocyte classes as well. Cured animals were rechallenged with J774 cells, and the tumors were rejected in 71% of mice. Cured mice had tumor-specific T cells in spleens as determined by a 51Cr release assay. We conclude that low-dose CY depletes T-regs and potentiates BPD–PDT, leading to tumor cures and memory immunity.

Impaired apoptosis, extended duration of immune responses, and a lupus-like autoimmune disease in IEX-1-transgenic mice

Susceptibility of activated T cells to apoptosis must be tightly regulated to ensure sufficient T cell progeny for an effective response, while allowing a rapid depletion of them at the end of the immune response. We show here that a previously isolated, NF-κB/rel target gene IEX-1 (Immediate Early response gene X-1) is highly expressed in T cells at early stages of activation, but declines with a prolonged period of activation time, coincident with an increased susceptibility of T cells to apoptosis during the late phases of an immune response. Transgenic expression of IEX-1 specifically in lymphocytes impaired apoptosis in activated T cells, extended a duration of an effector-phase of a specific immune response, and increased the accumulation of effector/memory-like T cells and the susceptibility to a lupus-like autoimmune disease. Our study demonstrated an antiapoptotic effect of IEX-1 on T cell apoptosis triggered by ligation of Fas and T cell receptor (TCR)/CD3 complex. The ability of extending life expectancy of T effectors, in line with a decrease in its expression following prolonged T cell activation, suggests a key role for IEX-1 in regulating T cell homeostasis during immune responses.

Changes in bone density and turnover after alendronate or estrogen withdrawal.

Objective: To compare bone mineral density (BMD) and bone turnover changes after therapy withdrawal in postmenopausal women treated with alendronate or estrogen-progestin. Design: In this randomized, blinded, multinational, placebo-controlled trial, 1,609 healthy postmenopausal women ages 45 to 59 years were assigned to receive alendronate, placebo, or open-label estrogen-progestin (conjugated equine estrogens plus medroxyprogesterone acetate or a cyclic regimen of 17β-estradiol, norethisterone acetate and estradiol). Of the original women, one third after year 2 and one third after year 4 were switched from alendronate to placebo, while remaining blinded to treatment assignment. The women taking estrogen-progestin in years 1 to 4 were followed off therapy in years 5 and 6. BMD at the lumbar spine and hip and biochemical markers of bone turnover were measured. Results: The treatment groups described in the current report represent 860 women at baseline; 481 women entered year 5, and 430 completed 6 years. BMD steadily decreased in the placebo group during all 6 years. In contrast, spine and hip BMD increased during the first 4 years in the groups receiving daily continuous alendronate 5 mg and estrogen-progestin. During years 5 and 6, BMD decreased at the lumbar spine -2.42% (95% CI = −4.10, −0.74) and total hip −1.09% (−2.60, 0.41) in the group previously treated with alendronate 5 mg for 4 years. In comparison, large BMD decreases were observed at the spine [−7.69% (−8.96, −6.41)] and total hip [−5.16% (−6.30, −4.01)] among women who had received estrogen-progestin for 4 years. Conclusion: Alendronate produces greater residual skeletal effects than estrogen-progestin after therapy discontinuation.

Photodynamic Therapy of Tumors Can Lead to Development of Systemic Antigen-Specific Immune Response

Background The mechanism by which the immune system can effectively recognize and destroy tumors is dependent on recognition of tumor antigens. The molecular identity of a number of these antigens has recently been identified and several immunotherapies have explored them as targets. Photodynamic therapy (PDT) is an anti-cancer modality that uses a non-toxic photosensitizer and visible light to produce cytotoxic reactive oxygen species that destroy tumors. PDT has been shown to lead to local destruction of tumors as well as to induction of anti-tumor immune response. Methodology/Principal Findings We used a pair of equally lethal BALB/c colon adenocarcinomas, CT26 wild-type (CT26WT) and CT26.CL25 that expressed a tumor antigen, β-galactosidase (β-gal), and we treated them with vascular PDT. All mice bearing antigen-positive, but not antigen-negative tumors were cured and resistant to rechallenge. T lymphocytes isolated from cured mice were able to specifically lyse antigen positive cells and recognize the epitope derived from beta-galactosidase antigen. PDT was capable of destroying distant, untreated, established, antigen-expressing tumors in 70% of the mice. The remaining 30% escaped destruction due to loss of expression of tumor antigen. The PDT anti-tumor effects were completely abrogated in the absence of the adaptive immune response. Conclusion Understanding the role of antigen-expression in PDT immune response may allow application of PDT in metastatic as well as localized disease. To the best of our knowledge, this is the first time that PDT has been shown to lead to systemic, antigen- specific anti-tumor immunity.

Functional interaction of DFF35 and DFF45 with caspase-activated DNA fragmentation nuclease DFF40.

DNA fragmentation factor (DFF) functions downstream of caspase-3 and directly triggers DNA fragmentation during apoptosis. Here we described the identification and characterization of DFF35, an isoform of DFF45 comprised of 268 amino acids. Functional assays have shown that only DFF45, not DFF35, can assist in the synthesis of highly active DFF40. Using the deletion mutants, we mapped the function domains of DFF35/45 and demonstrated that the intact structure/conformation of DFF45 is essential for it to function as a chaperone and assist in the synthesis of active DFF40. Whereas the amino acid residues 101–180 of DFF35/45 mediate its binding to DFF40, the amino acid residues 23–100, which is homologous between DFF35/45 and DFF40, may function to inhibit the activity of DFF40. In contrast to DFF45, DFF35 cannot work as a chaperone, but it can bind to DFF40 more strongly than DFF45 and can inhibit its nuclease activity. These findings suggest that DFF35 may function in vivo as an important alternative mechanism to inhibit the activity of DFF40 and further, that the inhibitory effects of both DFF35 and DFF45 on DFF40 can put the death machinery under strict control.

Endothelial dysfunction enhances vasoconstriction due to scavenging of nitric oxide by a hemoglobin-based oxygen carrier

Background:To date, there is no safe and effective hemoglobin-based oxygen carrier (HBOC) to substitute for erythrocyte transfusion. It is uncertain whether a deficiency of endothelial nitric oxide bioavailability (endothelial dysfunction) prevents or augments HBOC-induced vasoconstriction. Methods:Hemodynamic effects of infusion of PolyHeme (1.08 g hemoglobin/kg; Northfield Laboratories, Evanston, IL) or murine tetrameric hemoglobin (0.48 g hemoglobin/kg) were determined in awake healthy lambs, awake mice, and anesthetized mice. In vitro, a cumulative dose-tension response was obtained by sequential addition of PolyHeme or tetrameric hemoglobin to phenylephrine-precontracted murine aortic rings. Results:Infusion of PolyHeme did not cause systemic hypertension in awake lambs but produced acute systemic and pulmonary vasoconstriction. Infusion of PolyHeme did not cause systemic hypertension in healthy wild-type mice but induced severe systemic vasoconstriction in mice with endothelial dysfunction (either db/db mice or high-fat fed wild-type mice for 4–6 weeks). The db/db mice were more sensitive to systemic vasoconstriction than wild-type mice after the infusion of either tetrameric hemoglobin or PolyHeme. Murine aortic ring studies confirmed that db/db mice have an impaired response to an endothelial-dependent vasodilator and an enhanced vasoconstrictor response to HBOC. Conclusions:Reduction in low molecular weight hemoglobin concentrations to less than 1% is insufficient to abrogate the vasoconstrictor effects of HBOC infusion in healthy awake sheep or in mice with reduced vascular nitric oxide levels associated with endothelial dysfunction. These findings suggest that testing HBOCs in animals with endothelial dysfunction can provide a more sensitive indication of their potential vasoconstrictor effects.

Facilitation of transcutaneous drug delivery and vaccine immunization by a safe laser technology

Full-surface laser ablation has been shown to efficiently disrupt stratum corneum and facilitate transcutaneous drug delivery, but it is frequently associated with skin damage that hampers its clinic use. We show here that a safer ablative fractional laser (AFL) can sufficiently facilitate delivery of not only patch-coated hydrophilic drugs but also protein vaccines. AFL treatment generated an array of self renewable microchannels (MCs) in the skin, providing free paths for drug and vaccine delivery into the dermis while maintaining integrity of the skin by quick healing of the MCs. AFL was superior to tape stripping in transcutaneous drug and vaccine delivery as a much higher amount of sulforhodamine B (SRB), methylene blue (MB) or a model vaccine ovalbumin (OVA) was recovered from AFL-treated skin than tape-stripped skin or control skin after patch application. Following entry into the MCs, the drugs or OVA diffused quickly to the entire dermal tissue via the lateral surface of conical-shaped MCs. In contrast, a majority of the drugs and OVA remained on the skin surface, unable to penetrate into the dermal tissue in untreated control skin or tape stripping-treated skin. Strikingly, OVA delivered through the MCs was efficiently taken up by epidermal Langerhans cells and dermal dendritic cells in the vicinity of the MCs or transported to the draining lymph nodes, leading to a robust immune response, in sharp contrast to a weak, though significant, immune response elicited in tape stripping group or a basal immune response in control groups. These data support strongly that AFL is safe and sufficient for transcutaneous delivery of drugs and vaccines.

Distinct Domains for Anti- and Pro-apoptotic Activities of IEX-1

IEX-1 (immediate early response gene X-1) is a stress-inducible gene. Its overexpression can suppress or enhance apoptosis dependent on the nature of stress, yet the polypeptide does not possess any of the functional domains that are homologous to those present in well characterized effectors or inhibitors of apoptosis. This study using sequence-targeting mutagenesis reveals a transmembrane-like integrated region of the protein to be critical for both pro-apoptotic and anti-apoptotic functions. Substitution of the key hydrophobic residues with hydrophilic ones within this region impairs the capacity IEX-1 to positively and negatively regulate apoptosis. Mutations at N-linked glycosylation and phosphorylation sites or truncation of the C terminus of IEX-1 also abrogated its potential to promote cell survival. However, distinguished from the transmembrane-like domain, these mutants preserved pro-apoptotic activity of IEX-1 fully. On the contrary, mutation of nuclear localization sequence, despite its importance in apoptosis, did not impede IEX-1-mediated cell survival. Strikingly, all the mutants that lose their anti-apoptotic ability are unable to prevent acute increases in production of intracellular reactive oxygen species (ROS) at the initial onset of apoptosis, whereas those mutants that can sustain anti-death function also control acute ROS production as sufficiently as wild-type IEX-1. These findings suggest a critical role of IEX-1 in regulation of intracellular ROS homeostasis, providing new insight into the mechanism underlying IEX-1-mediated cell survival.

A Novel Laser Vaccine Adjuvant Increases the Motility of Antigen Presenting Cells

Background Development of a potent vaccine adjuvant without introduction of any side effects remains an unmet challenge in the field of the vaccine research. Methodology/Principal Findings We found that laser at a specific setting increased the motility of antigen presenting cells (APCs) and immune responses, with few local or systemic side effects. This laser vaccine adjuvant (LVA) effect was induced by brief illumination of a small area of the skin or muscle with a nondestructive, 532 nm green laser prior to intradermal (i.d.) or intramuscular (i.m.) administration of vaccines at the site of laser illumination. The pre-illumination accelerated the motility of APCs as shown by intravital confocal microscopy, leading to sufficient antigen (Ag)-uptake at the site of vaccine injection and transportation of the Ag-captured APCs to the draining lymph nodes. As a result, the number of Ag+ dendritic cells (DCs) in draining lymph nodes was significantly higher in both the 1° and 2° draining lymph nodes in the presence than in the absence of LVA. Laser-mediated increases in the motility and lymphatic transportation of APCs augmented significantly humoral immune responses directed against a model vaccine ovalbumin (OVA) or influenza vaccine i.d. injected in both primary and booster vaccinations as compared to the vaccine itself. Strikingly, when the laser was delivered by a hair-like diffusing optical fiber into muscle, laser illumination greatly boosted not only humoral but also cell-mediated immune responses provoked by i.m. immunization with OVA relative to OVA alone. Conclusion/Significance The results demonstrate the ability of this safe LVA to augment both humoral and cell-mediated immune responses. In comparison with all current vaccine adjuvants that are either chemical compounds or biological agents, LVA is novel in both its form and mechanism; it is risk-free and has distinct advantages over traditional vaccine adjuvants.