Safety and acceptability of intravaginal rings releasing estradiol and progesterone.

OBJECTIVE: This study aimed to evaluate the safety and acceptability of two fixed-dose 28-day vaginal ring formulations of 17ß-estradiol (E2) and progesterone (P4) to treat vasomotor symptoms (VMS) and the genitourinary syndrome of menopause. DESIGN: DARE HRT1-001 was the first-in-woman study of 28-day exposure to two 28-day intravaginal rings (IVRs) designed to release 80 µg/day E2 + 4 mg/day P4 (IVR1) or 160 µg/day E2 + 8 mg/day P4 (IVR2) compared with oral E2 1 mg/day + oral P4 100 mg/day. To assess safety, participants completed a daily diary to record treatment emergent adverse events (TEAEs). To determine acceptability, at the end of treatment IVR users completed a questionnaire assessing tolerability and usability. RESULTS: Enrolled women (n = 34) were randomized to use IVR1 (n = 10), IVR2 (n = 12) or oral (n = 12). Thirty-one participants (IVR1 = 10, IVR2 = 10, oral = 11) completed the study. The TEAE profile of those in the IVR groups were similar to the referent oral regimen. TEAEs related to the study product were more common with IVR2 use. Endometrial biopsies were not performed unless endometrial thickness was >4 mm or for clinically significant postmenopausal bleeding. One IVR1 participant had an endometrial stripe increase from 4 mm at screening to 8 mm at the end of treatment. The biopsy indicated no evidence of plasma cells or endometritis and no evidence of atypia, hyperplasia or malignancy. Two other endometrial biopsies were performed for postmenopausal bleeding with similar findings. There were no clinically meaningful laboratory or vital sign abnormalities or trends identified in observed values or changes from baseline. Pelvic speculum examination identified no clinically significant abnormalities in any participant at any visit. Tolerability and usability data demonstrated that both IVRs were generally highly acceptable. CONCLUSIONS: Both IVR1 and IVR2 were safe and well tolerated in healthy postmenopausal women. TEAE profiles were comparable to the referent oral regimen.

Description and comparison of brain distribution of oxytocin receptors in Rhabdomys pumillio and Rhabdomys dilectus.

Oxytocin receptor (OXTR) distribution in the brain has been associated with different reproductive and social strategies of species. Rhabdomys pumilio (R. pumilio) and Rhabdomys dilectus (R. dilectus) are two sister rodent species that live in large/medium (but flexible) or small (mostly solitary) social groups respectively. In this study, we describe and compare the distribution of OXTR in these two species. OXTR binding in the brain of R. pumilio (8 females and 5 males) and R. dilectus (8 females and 5 males) adults was determined using autoradiography. Our results revealed significant differences in the nucleus accumbens, diagonal band, medial preoptic area, lateral habenula, superior colliculus, periaqueductal area and anterior paraventricular nucleus (higher in R. dilectus), and the dorsal lateral septum and anterior bed nucleus of the stria terminalis (higher in R. pumilio). OXTR density in other brain regions, such as the amygdala nuclei and hippocampus, did not differ between the two species. Sex differences were found in the medial preoptic area and ventral region of the lateral septum in R. pumilio (OXTR density higher in males) and in the anterior paraventricular thalamic nucleus, ventromedial nucleus of the hypothalamus and basolateral amygdala of R. dilectus (OXTR density higher in females). A sex difference in the density of OXTR was also found in the posterior region of the bed nucleus of the stria terminalis, where it was higher in males than in females of both species. This study shows species-specific brain distribution of OXTR in R. pumilio and R. dilectus that are unique, but with similarities with other polygynous or promiscuous rodent species that live in variable size groups, such as R. norvergicus, C. sociabilis, S. teguina and M. musculus.

WHY WOULDN'T I JOIN IMS?.

in January, I had the pleasure of speaking at the annual meeting of the Scott County Medical Society (SMCS), my home county and local medical society. It was great to see colleagues and friends I've known for many years, but equally nice was meeting new physicians and their spouses. Several physicians, including one of my partners, were honored for 25 consecutive years of commitment and service to SCMS. In another time, a continuous 25 year commitment to organized medicine was common; not only was membership a way to stay connected and involved in the medical community, it was a privilege of our profession. Times have changed and many physicians no longer feel that sense of professional responsibility to organized medicine. I can't help but ponder the impact on our medical society - both at the state and county levels - the cost to our profession, and the reasons behind the apathy.

Development of temperature correction equations for bioelectrical impedance analysis models for brook trout Salvelinus fontinalis.

The objective of this study was to establish the relationships between bioelectrical impedance analysis (BIA) measures (resistance and reactance) and temperature and to determine if corrections improve BIA models for brook trout Salvelinus fontinalis when used over a wide range of temperatures. Both resistance and reactance significantly decreased as temperature increased. Application of temperature corrections to BIA models attempting to predict per cent dry mass reduced root-mean-squared error by an average of 32%. Researchers taking BIA measures on fishes in the field where temperature varies will need to correct resistance and reactance to the temperature at which the BIA model was developed for successful predictions of per cent dry mass to be possible. This study presents a clear description of methods that can be used to developed temperature correction equations so that future researchers can use BIA in any field setting and increase the accuracy of BIA-based estimates of per cent dry mass.

T cell receptor usage and fine specificity of human immunodeficiency virus 1-specific cytotoxic T lymphocyte clones: analysis of quasispecies recognition reveals a dominant response directed against a minor in vivo variant.

Numerous virus-specific, class I-restricted cytotoxic T lymphocyte (CTL) epitopes have been identified, yet little information is available regarding the specificity of the CTL response in persons of the same human histocompatibility leukocyte antigen (HLA) type. In this study, the human immunodeficiency virus (HIV) 1 envelope-specific CTL response was evaluated in five HLA-B14-positive persons. CTL responses specific for a previously described nine-amino acid epitope in gp41 (aa 584-592, ERYLKDQQL) could be identified in all subjects, and CTL clones specific for this epitope could be isolated from four persons. Despite heterogeneous T cell receptor usage, the fine specificity of the clones was similar, as defined by recognition of alanine-substituted peptides as well as peptides representing natural HIV-1 sequence variants. Correlation with in vivo virus sequences revealed that the dominant species in two of the subjects represented poorly recognized variants, with a K-->Q substitution at amino acid 588, whereas no variants were observed in the other two subjects. Although clonal type-specific responses to these dominant variants could be identified, the magnitude of these responses remained small, and the dominant CTL response was directed at the minor in vivo variant. These studies indicate that despite similar epitope-specific immunologic pressure in persons of the same HLA type, the in vivo quasispecies may differ, and that the major in vivo immune response to a given CTL epitope can be directed at a minor variant.

A novel HLA-A*03 allele, HLA-A*03:71.

A novel HLA-A*03 allele, HLA-A*03:71, was identified by PCR sequence-based typing.

Structure of RNA in ribosomes.

The 50S and 30S ribosomes and 23S and 16S RNA were hydrolyzed with ribonuclease A. The rate constants and number of fragments produced were determined for each reaction. The conformation of 23S RNA changes when the RNA is extracted from the ribosome. Specific regions of the RNA in 50S and 30S ribosomes are protected from hydrolysis by the ribosomal proteins.

Secondary structure of ribosomal RNA.

Infrared spectra were obtained for 16S and for 23S ribosomal RNA's in D(2)O solutions. The percentage of each base in the paired and unpaired regions of the RNA was determined from the spectra. The secondary structures of 16S and 23S ribosomal RNA's (from Escherichia coli) are significantly different from each other and are also different from those of yeast ribosomal RNA, formylmethionyl-transfer RNA, and the anticodon fragment of this transfer RNA.

A preliminary operational classification system for nonmutagenic modes of action for carcinogenesis.

This article proposes a system of categories for nonmutagenic modes of action for carcinogenesis. The classification is of modes of action rather than individual carcinogens, because the same compound can affect carcinogenesis in more than one way. Basically, we categorize modes of action as: (1) co-initiation (facilitating the original mutagenic changes in stem and progenitor cells that start the cancer process) (e.g. induction of activating enzymes for other carcinogens); (2) promotion (enhancing the relative growth vs differentiation/death of initiated clones (e.g. inhibition of growth-suppressing cell-cell communication); (3) progression (enhancing the growth, malignancy, or spread of already developed tumors) (e.g. suppression of immune surveillance, hormonally mediated growth stimulation for tumors with appropriate receptors by estrogens); and (4) multiphase (e.g., "epigenetic" silencing of tumor suppressor genes). A priori, agents that act at relatively early stages in the process are expected to manifest greater relative susceptibility in early life, whereas agents that act via later stage modes will tend to show greater susceptibility for exposures later in life.

Lack of strong immune selection pressure by the immunodominant, HLA-A*0201-restricted cytotoxic T lymphocyte response in chronic human immunodeficiency virus-1 infection.

Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.

Efficient generation of human T cells from a tissue-engineered thymic organoid.

Biocompatible inorganic matrices have been used to enhance bone repair by integrating with endogenous bone architecture. Hypothesizing that a three-dimensional framework might support reconstruction of other tissues as well, we assessed the capacity of a tantalum-coated carbon matrix to support reconstitution of functioning thymic tissue. We engineered a thymic organoid by seeding matrices with murine thymic stroma. Co-culture of human bone marrow-derived hematopoietic progenitor cells within this xenogeneic environment generated mature functional T cells within 14 days. The proportionate T-cell yield from this system was highly reproducible, generating over 70% CD3+ T cells from either AC133+ or CD34+ progenitor cells. Cultured T cells expressed a high level of T-cell receptor excision circles (TREC), demonstrating de novo T lymphopoiesis, and function of fully mature T cells. This system not only facilitates analysis of the T-lymphopoietic potential of progenitor cell populations; it also permits ex vivo genesis of T cells for possible applications in treatment of immunodeficiency.

Cytokinetic and biochemical effects of 5-iminodaunorubicin in human colon carcinoma in culture.

The semisynthetic anthracycline, 5-iminodaunorubicin (IM), was investigated to see whether modification of the benzoquinone moiety to produce a drug with low free radical potential would alter the cytotoxic and biochemical characteristics of this drug in comparison to Adriamycin (ADR), an agent with high free radical potential. Cell viability was measured in human colon carcinoma (HT-29) cells by soft-agar cloning. Upon exposure to either log-phase or early plateau-phase cells for 2 hr to IM or ADR, a threshold exponential cell lethality curve was obtained. Prolonging during exposure to 24 hr produced an exponential decline in cell survival and a marked reduction in viability of both log-phase and early plateau-phase cells. Inhibition of DNA synthesis in log-phase cells after 2 and 24 hr of exposure to IM and ADR paralleled the increased cell lethality produced by the drugs. In contrast, total RNA synthesis was not inhibited by IM, whereas ADR impaired both RNA and DNA synthesis. Nuclear rRNA, synthesis was not significantly inhibited following 24 hr of exposure to 10(-7) M ADR or IM but was inhibited by 85 and 35% at 10(-6) M ADR or IM, respectively. The affinity of IM and ADR for HT-29 DNA was measured in vitro by displacement of acridine orange binding and was found to be similar for both analogs. These studies suggest that the cytotoxicity of IM and ADR results from the interactions of these drugs with DNA.

Lymphatic absorption and tissue disposition of liposome-entrapped [14C]adriamycin following intraperitoneal administration to rats.

The lymphatic absorption and tissue distribution of free [14C]Adriamycin, "empty" [3H]liposomes, free [14C]Adriamycin plus empty [3H]liposomes, and [14C]Adriamycin entrapped into [3H]liposomes have been examined at intervals after i.p. injection into rats. Following treatment with empty [3H]liposomes, almost 30% of the liposomal lipid marker was recovered in 24-hr thoracic duct lymph, but when [14C]Adriamycin was added to or encapsulated in liposomes, this value was reduced to 10%. Conversely, only 1% of free [14C]Adriamycin was recovered in 24-hr lymph, but liposomal encapsulation produced a six-fold increase in this value. Studies on the tissue distribution of the liposomal lipid marker after dosing with empty liposomes revealed uptake by diaphragm, liver and spleen, but the highest tissue concentrations were noted in lymph nodes. Liposomal encapsulation of Adriamycin altered its tissue disposition, chiefly by increasing the concentration of drug equivalents in diaphragm, liver and spleen. Although free Adriamycin was accumulated by lymph nodes to some extent, this lymph node accumulation was markedly enhanced by liposomal encapsulation and was present only in those nodes through which lymph draining the peritoneal cavity passes. This finding, together with the observation that diaphragm and thoracic duct lymph contain relatively high levels of liposomal lipid and Adriamycin equivalents, indicates that liposomes are selectively absorbed from the peritoneal cavity by lymphatics and are retained by certain lymph nodes. The results of this study suggest that i.p. administration of liposome-encapsulated drugs may provide a means of selectively concentrating anti-tumor agents in lymphatic channels and lymph nodes.

Role of protein kinase C in phosphorylation of vinculin in adriamycin-resistant HL-60 leukemia cells.

In response to phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), HL-60 cells differentiate to macrophage-like cells and exhibit the ability to phosphorylate vinculin in vitro. Adriamycin-resistant HL-60 (HL-60/ADR) cells similarly demonstrate this characteristic without prior treatment with TPA. Since protein kinase C (PK-C) is a cellular TPA receptor, we have examined the role of this enzyme in the inherent ability of HL-60/ADR cells to phosphorylate vinculin. DEAE-cellulose chromatography of cell extracts revealed that HL-60/ADR cells contained 2-fold more PK-C than did the parental cell line. All PK-C activity was found in the cytosol of wild type HL-60 cells, whereas 85% of PK-C activity was cytosolic and 15% was membrane-bound in HL-60/ADR cells. After a 2-day treatment with 10 nM TPA, PK-C activity was reduced 80-90% in both cell lines regardless of its intracellular distribution. Immunoblotting of cell extracts from HL-60/ADR cells or HL-60 cells following treatment with TPA revealed increased levels of a 52-kDa species of similar mass to M-kinase. Coincident with these changes after TPA treatment was a reduction in Ca2+ and phospholipid-independent phosphorylation of vinculin in vitro in extracts from HL-60/ADR cells, whereas HL-60 cells exhibited an elevation of this phosphoprotein. The phosphorylation of vinculin in TPA-treated HL-60 cells or untreated HL-60/ADR cells was blocked by antibodies to protein kinase C. These results suggest that it is not the absolute level of protein kinase C but rather the proteolytic activation of PK-C to a Ca2+ and phospholipid-independent form which is associated with the utilization of vinculin as an endogenous substrate.

Superparamagnetic gadonanotubes are high-performance MRI contrast agents.

We report the nanoscale loading and confinement of aquated Gd3+n-ion clusters within ultra-short single-walled carbon nanotubes (US-tubes); these Gd3+n@US-tube species are linear superparamagnetic molecular magnets with Magnetic Resonance Imaging (MRI) efficacies 40 to 90 times larger than any Gd3+-based contrast agent (CA) in current clinical use.

Identification and treatment of metabolic abnormalities in patients with vertigo.

Hyperinsulinism, impaired glucose tolerance, and hypertriglyceridemia may be risk factors for atherosclerotic heart disease and have also been described in patients with vertigo, whose symptoms and findings responded to appropriate dietary therapy. We studied 100 patients in an otolaryngology practice to determine the role of these abnormalities in identifying patients suitable for dietary therapy and to assess the efficacy of dietary therapy in the treatment of vertigo in such selected patients. The determination of hyperinsulinism and hypertriglyceridemia were of value as supplements to the traditional glucose tolerance test in detecting reversible metabolic vertigo. Reactive hypoglycemia was found in only four patients and thus appears overdiagnosed as a cause of vertigo. Insulin resistance appears to be the basic abnormality in this syndrome, which, in our series, occurred predominantly in overweight patients.

Characterization of a helix-loop-helix (EF hand) motif of silver hake parvalbumin isoform B.

Parvalbumins are a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF-hand. Silver hake parvalbumin (SHPV) consists of three EF-type helix-loop-helix regions, two of which have the ability to bind calcium. The three helix-loop-helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV-B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a beta-lineage parvalbumin. SHPV-B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33-amino acid EF fragment (residues 76-108), containing one of the calcium ion binding sites in native SHPV-B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion-induced self-association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge-density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer-dimer equilibrium when complexed with La3+.