Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells.

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ERα) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ERα ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ERα breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29's activities in breast cancer cells harboring Y537S ERα. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells.

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ERα) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ERα ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ERα breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29's activities in breast cancer cells harboring Y537S ERα. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

Case work guidelines and interpretation of short tandem repeat complex mixture analysis.

Interpretation guidelines for short tandem repeat casework analysis are difficult to construct. As soon as a set of guidelines are developed, a new case evolves that does not fit the painstakingly written document. The casework analysts gather and amend the guidelines again, and again. This article seeks to demonstrate that general guidelines can be set and written such that it can be used for any detection format. Guidelines published by the Scientific Working Group for DNA Analysis Methods, a working group of DNA forensic experts in the United States, are used to set the format for the written protocol on interpretations. The rule "the interpretation of results in casework is a matter of professional judgment and expertise. Not every situation can or should be covered by a preset rule" is stressed. Development of minimum and maximum threshold values, heterozygote ratios, stochastic limits, and determination of major and minor components based on validation studies is discussed. The paper travels through setting criteria to evaluate internal lane standards and amplification controls. It continues with establishing ranges for interpretation and defining true alleles versus anomalies. Examples of a variety of profiles are given and the potential interpretation, using signal intensities and genetics. In addition, report writing strategies and wording routinely used by the Pennsylvania State Police DNA Laboratory System are given.

Correlation between scanning laser ophthalmoscope microperimetry and anatomic abnormalities in patients with subfoveal neovascularization.

PURPOSE: The purpose of the study is to identify the anatomic abnormalities associated with an absolute scotoma and the location and stability of fixation in patients with subfoveal neovascularization in age-related macular degeneration, presumed ocular histoplasmosis syndrome, and other disorders. METHODS: Scanning laser ophthalmoscope microperimetry was superimposed on color fundus photographs and fluorescein angiograms of 21 eyes with subfoveal neovascular membranes secondary to age-related macular degeneration (14 eyes) and presumed ocular histoplasmosis syndrome (7 eyes). The authors determined the location and the area occupied by the absolute scotoma and each of the following subretinal lesions: subretinal hemorrhage, neurosensory retinal detachment, retinal pigment epithelial (RPE) atrophy, RPE hyperplasia, atrophy of the choriocapillaris, hard exudates, and the subfoveal neovascular membrane. The area of absolute scotoma determined by scanning laser ophthalmoscope microperimetry was superimposed on the anatomic lesions. The authors calculated the relative risk ratio (RR) of an absolute scotoma occurring in regions corresponding to each anatomic abnormality, and determined the preferred location and stability of fixation in each eye. RESULTS: An absolute scotoma was present in areas of chorioretinal scar (RR = 107.61), RPE atrophy (RR = 9.97), subretinal hemorrhage (RR = 2.88), and the neovascular membrane (RR = 1.86). Fixation was stable in all patients with presumed ocular histoplasmosis syndrome but only 29% of patients with age-related macular degeneration. Fifty-five percent of patients with stable fixation fixated over an area of RPE hyperplasia. CONCLUSION: The relative risk of an absolute scotoma is highest over areas of chorioretinal scars, RPE atrophy, subretinal hemorrhage, and the neovascular membrane. Fixation is more stable in patients with subfoveal neovascularization from presumed ocular histoplasmosis syndrome than with age-related macular degeneration and frequently is present over an area of RPE hyperplasia.

Screening corneas for human immunodeficiency virus type 1 proviral DNA by polymerase chain reaction.

PURPOSE: We evaluated the sensitivity of the polymerase chain reaction as a technique to directly screen potential donor corneas for human immunodeficiency virus type 1 (HIV-1) proviral DNA. METHODS: DNA from the central 8.0-mm cornea, limbal cornea, aqueous humor, and retina from 22 eyes of 11 cadavers seropositive for HIV was extracted and amplified by polymerase chain reaction using primers specific for the gag and env regions of the HIV-1 genome. The identity of amplification products was confirmed by Southern blot hybridization. RESULTS: Viral DNA was detected in four (18.2%) of 22 central corneas, one (4.5%) of 22 limbal corneas, one (6.3%) of 16 aqueous humor samples, and seven (31.8%) of 22 retinas. No correlation was noted between the presence of HIV-1 proviral DNA in samples from the central cornea and from the other tissues tested from the same eye. CONCLUSIONS: Within the limits of our assay, processing and analysis of limbal cornea, aqueous humor, and retina by polymerase chain reaction may not reliably ascertain the presence of HIV-1 in the central, transplantable cornea.

Immunohistochemical localization of fibrillin in human ocular tissues. Relevance to the Marfan syndrome.

OBJECTIVE: To better understand the ocular manifestations of the Marfan syndrome, we investigated the distribution of fibrillin in normal human ocular tissues. Fibrillin, a microfibrillar glycoprotein component of the extracellular matrix, has been found to be the defective gene product in the Marfan syndrome. METHODS: Frozen sections from seven pairs of normal eyes were stained with mouse anti-human fibrillin antibodies using the avidin-biotin immunoperoxidase technique. RESULTS: In the anterior segment, the following exhibited positive staining for fibrillin: the lens capsule and zonules; connective tissues of the iris, ciliary body, ciliary processes, and conjunctiva; and the basement membrane regions of the corneal epithelium and endothelium of Schlemm's canal. Posteriorly, fibrillin localized to the lamina cribrosa, sclera, choroid, and Bruch's membrane. CONCLUSIONS: Fibrillin is widely distributed in ocular connective tissues. The implications of defects in these tissues and the resultant ocular abnormalities in the Marfan syndrome such as ectopia lentis and glaucoma are discussed.

Comparative diagnosis of neonatal chlamydial conjunctivitis by polymerase chain reaction and McCoy cell culture.

Cervical and ocular swabs from 100 mother/newborn pairs delivering on the clinic service were assayed for Chlamydia trachomatis with standard McCoy cell culture and with standard and biotinylated polymerase chain reaction techniques, using primers directed against the major outer membrane protein gene and C. trachomatis-specific cryptic plasmid, respectively. Using the polymerase chain reaction, 20 (20%) mothers and seven (7%) neonates were positive for Chlamydia. All neonates positive by polymerase chain reaction were from mothers positive by polymerase chain reaction, yielding a 35% transmission rate. Only five of 20 (25%) mothers and two of seven (28%) neonates positive by polymerase chain reaction were positive by cell culture. All cell culture samples were positive by polymerase chain reaction testing. Culture and polymerase chain reaction analysis two weeks after treatment with oral erythromycin were negative. The polymerase chain reaction assay appears to be equally specific and more sensitive than McCoy cell culture for the detection of C. trachomatis from ocular specimens.

Herpesvirus antibody levels in the etiologic diagnosis of the acute retinal necrosis syndrome.

Quantitative antibody levels to three herpesviruses in acute and chronic sera from six patients with clinical signs of the acute retinal necrosis syndrome were consistent with a specific etiologic diagnosis only in the two cases associated with cutaneous herpes zoster. Available data on acute and convalescent antibody titers to herpes group viruses from these six patients in addition to data from 27 acute retinal necrosis cases from the literature disclosed that only 13 of the 33 patients (39%) had a diagnostic increase or decrease in herpes group viral antibody levels on serial sampling. Three patients had nondiagnostic changes in viral antibody levels despite positive vitreous cultures for herpesviruses. In contrast, a review of 25 cases from the literature with paired antiviral serum and intraocular fluid antibody levels suggested a more promising approach to the etiologic diagnosis of the acute retinal necrosis syndrome. By calculating the ratio of antiviral antibodies in intraocular fluid and serum, an etiologic diagnosis could be made in 12 of 14 (86%) of subacute and convalescent samples. The sensitivity of this method decreased to 72% (13 of 18) when fluids were obtained earlier in the course of the disease.

Effects of human chorionic gonadotrophin on contractile activity of steroid-primed pig myometrium in vitro.

We examined the effects of (a) oestrogen and progesterone on concentrations of luteinizing hormone/human chorionic gonadotrophin (LH/hCG) receptors in uterine smooth muscle in vivo and (b) hCG on spontaneous myometrial contractions in vitro. Ovariectomized gilts received 2 ml corn oil (control; n = 5), 2 mg oestradiol benzoate (n = 6) or 20 mg progesterone (n = 5) for 5 days. Gilts were hysterectomized 8 h after the last injection and longitudinal sections of myometrium were incubated in modified Krebs' solution with 0 or 10 i.u. of hCG (n = 10/gilt) for 4 h at 37 degrees C in 95% O2:5% CO2. After incubation, myometrial sections were placed in a tissue chamber perfused with Krebs' solution and mechanical activity was recorded for 30 min. Cell membrane fractions were prepared from myometrial tissue not used for in-vitro studies and analysed for LH/hCG receptors. Treatment with oestradiol benzoate increased (P less than 0.01) the number of LH/hCG-binding sites compared with gilts receiving corn oil or progesterone. Incubation of myometrium with hCG reduced (P less than 0.01) the frequency and amplitude of spontaneous uterine contractions in gilts treated with oestradiol benzoate. In contrast, hCG had no effect (P greater than 0.05) on the pattern of myometrial contractions in gilts given corn oil or progesterone. These results indicate that oestradiol promotes the synthesis of LH/hCG receptors in pig myometrium and incubation of oestrogen-primed tissue with hCG has a quiescent effect on myometrial contractility.

Episodic secretion of gonadotrophins and ovarian steroids in jugular and utero-ovarian vein plasma during the follicular phase of the oestrous cycle in gilts.

Blood samples were collected simultaneously from the jugular and utero-ovarian veins of 13 gilts from Days 11 through 16 of the oestrous cycle. A luteolytic dose (10 mg) of PGF-2 alpha was given on Day 12 to facilitate the natural occurrence of luteolysis and standardize the associated decrease in concentrations of progesterone. The mean interval from PGF to oestrus was 5.5 +/- 0.7 days (mean oestrous cycle length = 17.5 +/- 0.7 days). Mean concentrations, pulse amplitudes and pulse frequencies of oestradiol and progesterone were greater (P less than 0.05) in the utero-ovarian than jugular vein. Secretory profiles of LH and FSH were similar (P greater than 0.05) in plasma collected simultaneously from both veins. Based on these data, temporal relationships among hormonal patterns of FSH and LH in the jugular vein and oestradiol and progesterone in the utero-ovarian vein were examined. Concentrations of progesterone declined (P less than 0.05) between Days 12 and 14, while all secretory variables for oestradiol increased (P less than 0.05) from Day 12 through 16 of the oestrous cycle. The pulsatile secretion of FSH remained relatively constant during the experiment. However, both pulse amplitude and mean concentration tended (P less than 0.2) to be lower on Day 16 compared with Day 12. The episodic secretion of LH shifted from a pattern characterized by high-amplitude, low-frequency pulses to one dominated by numerous pulses of diminishing magnitude between Days 13 and 14. From Days 14 to 16 of the oestrous cycle, 91% of all oestradiol pulses were temporally associated with gonadotrophin pulses composed of both FSH and LH episodes. However, pulses of oestradiol (52%) not associated with an episode of LH and/or FSH were observed on Days 12 and 13. These data demonstrate that during the follicular phase of the pig oestrous cycle substantial oestradiol production occurred coincident with luteolysis and before the shift in the episodic secretion of LH. The pool of follicles which ovulated was probably the source of this early increase in the secretion of oestradiol. Therefore, we propose that factors in addition to FSH and LH are involved in the initial selection of follicles destined to ovulate during the early stages of the follicular phase of the pig oestrous cycle. In contrast, high-frequency, low-amplitude pulses composed of LH and FSH were the predominant endocrine signal associated with oestradiol secretion during the second half of the oestrous cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Localized antigen challenge of the nasal mucosa.

We describe a localized nasal antigen challenge and the measurement of mediators found at the same site. Eight ragweed-allergic subjects were challenged on 2 days, 1 week apart. Challenges consisted of six sequential provocations, beginning with two control challenges (diluent for antigen-phenol-buffered saline) followed by four increasing antigen doses (0.6, 6, 60, and 160 protein nitrogen units) (antigen day) or an additional four control challenges (control day). The number of sneezes and the symptom scores increased significantly with increasing antigen doses. The levels of histamine and N-alpha-tosyl-L-arginine methyl ester-esterase activity increased in the eluted secretions on the antigen day, but not on the control day. The amount of secretions collected also increased per unit of time on the antigen day. We found no significant increase in the concentration level of either histamine or N-alpha-tosyl-L-arginine methyl ester-esterase in nasal secretions on either day. We conclude that the total amount of histamine and N-alpha-tosyl-L-arginine methyl ester-esterase activity increased per unit of time while their concentration did not.

Ruptured hepatic adenoma. A spectrum of presentation and treatment.

Six cases of ruptured hepatic adenoma treated in our medical center were reviewed with attention directed toward presenting symptomatology and methods of treatment. These patients, five women who were long-term users of oral contraceptives and one man who had never taken steroid medication, presented with right upper quadrant abdominal pain of variable degree and duration. The cardiovascular status of these patients was also variable, ranging from a normal blood pressure, which allowed an orderly workup, and planned resection of the tumor to hypovolemic shock requiring emergency laparotomy for control of hemorrhage. The extent of surgery depended on the location and the number of adenomas, with the goal being to resect the adenoma and control hemorrhage while preserving as much normal liver parenchyma as possible. The treatment of choice in this disease is resection of the tumor with a margin of normal liver parenchyma. In those cases in which that is not practical, resectional debridement has proven to be an effective alternative.

Use of the pulsed dye laser and ultrasonic lithotriptor for removal of multiple intrahepatic gallstones.

Two established urologic modalities used for removal of kidney stones were applied in a complementary manner for percutaneous transhepatic removal of multiple intrahepatic gallstones. A pulsed dye laser was used through a flexible nephroscope to fragment a large impacted stone. A stone basket was used to remove stone fragments and a few small entrapped stones. Subsequently, an ultrasonic lithotriptor carried by a rigid nephroscope was tried and found to be a much more efficient means of removing the remaining small stones.

Alterations in gonadotropin secretion and ovarian function in prepubertal gilts by elevated environmental temperature.

The effect of chronic exposure to elevated environmental temperature on gonadotropin secretion and ovarian function was studied in prepubertal gilts. Gilts were maintained under control (15.6 degrees C) or elevated temperature (33.3 degrees C) conditions from 150 to 180 days of age. Endocrine and ovarian responses to bilateral (BLO), unilateral (ULO), and sham ovariectomy were evaluated between 175 and 180 days of age. During the 96-h sampling period after BLO, plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were suppressed in heat-stressed females. Similarly, elevated temperatures abolished the transient rise in FSH and subsequent follicular growth normally associated with ULO. In contrast, environmental treatment had no effect on the secretion of FSH and LH after sham ovariectomy, yet the number of small follicles was lower in gilts exposed to elevated temperatures than in females maintained under control conditions. These results indicate that a chronic exposure to elevated environmental temperature during pubertal development diminished the ability of the hypothalamo-hypophyseal axis to secrete FSH and LH, which had physiological consequences on follicular growth. When provided an appropriate stimulus (ULO), an acute period of FSH secretion and subsequent development of follicles failed to occur in females exposed to elevated temperatures. Consequently, we propose that delayed puberty in gilts during periods of elevated environmental temperatures is due, in part, to a diminished capacity for gonadotropin secretion.

The effect of a leukotriene antagonist on the early response to antigen.

The sulfidopeptide leukotrienes (LT) C4, D4, and E4 increase in nasal secretions during the nasal response to antigen challenge, and nasal challenge with LTD4 induces nasal congestion. To further assess the role of leukotrienes, we administered an oral LTD4 antagonist, L-649,923, to 12 patients who had nasal allergy to grass and ragweed pollen, in a double-blind, placebo-controlled, crossover study. Patients were challenged intranasally with increasing doses of pollen on each of 2 days, and the recovered nasal lavage fluids were assayed for histamine, TAME-esterase activity, and immunoreactive LTC4/D4/E4. The patients graded runny nose, congestion, and throat irritation, and sneezes were counted. Significant (p less than 0.01) increases in all parameters were found when comparing antigen challenge with diluent challenge, but no differences were seen among the treatment groups. Thus, oral L-649,923 was not effective in blocking the symptoms of the early nasal allergic response.

Endocrine changes associated with a dietary-induced increase in ovulation rate (flushing) in gilts.

Effects of an increased level of dietary energy (flushing) on plasma concentrations of FSH, LH, insulin, progesterone and estradiol-17 beta and ovulation rate were studied in 16 gilts. Gilts received 5,400 kcal ME/d for one estrous cycle and the first 7 d of a second. On d 8 of the second estrous cycle, gilts received either 5,400 kcal ME/d (control [C], n = 8) or 11,000 kcal ME/d (flushed [F], n = 8) for the remainder of the estrous cycle. Blood was collected daily at 15-min intervals for 6 h from d 8 through estrus. Gilts were examined by laparotomy 6 d after estrus. Ovulation rate was greater (P less than .05) in F than C gilts (16.0 vs 9.4). Mean daily concentrations of FSH were greater (P less than .05) in F gilts at 5 d, 4 d and 3 d prior to estrus compared with C females. In both C and F gilts, FSH decreased (P less than .05) prior to estrus. Mean daily concentrations of LH and LH pulse amplitude were not different (P greater than .05) between treatments. Mean number of LH pulses/6 h at 4 d, 3 d and 2 d prior to estrus were greater (P less than .05) in F than in C gilts. In both treatments, LH pulse amplitude decreased (P less than .05) and pulse frequency increased (P less than .07) prior to estrus. Mean plasma concentrations of insulin tended to be higher (P less than .07) in F than in C females during the 7-d period before estrus.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of elevated ambient temperatures on puberty in gilts.

Effects of elevated ambient temperature on puberty and related physiological responses were studied in 40 gilts. Group 1 (n = 20) gilts were born in September and Group 2 (n = 20) gilts were born in March. Gilts were placed in environmentally controlled chambers at 140 d of age. After a 10-d acclimation period at 20 degrees C, 35% relative humidity (RH), and 12 h light (L)/12 h dark (D), gilts within each group were randomly assigned to one of two treatments: control (C; 15.6 degrees C, 35% RH, 12 h L/12 h D) or heat stress (HS; 33.3 degrees C, 35% RH, 12 h L/12 h D). Daily detection of estrus with a boar began at 180 d of age and continued for 50 d. All gilts not reaching puberty by 230 d of age received 1,000 IU pregnant mare serum gonadotropin (PMSG) and 7 d later were examined by laparotomy. Rectal temperatures (REC) and respiration rates (RESP) were taken twice daily. Food intake (FI) and water usage (WC) were recorded daily. Blood samples were collected weekly and BW recorded at 150, 190, and 230 d of age. No differences (P greater than .05) were observed due to season of birth. Heat-stressed gilts had greater (P less than .001) REC and RESP and consumed more (P less than .01) water than C gilts. Food intake and ADG were not different between treatments (P greater than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of effects of zearalenone and estradiol benzoate on reproductive function during the estrous cycle in gilts.

Effects of estradiol benzoate (EB) and zearalenone (Z) on luteal maintenance and plasma hormone concentrations were studied in 45 gilts. Gilts were allocated to receive either 20 mg Z, 2 mg EB or no treatment (C) on d 1 to 5 (T1), 6 to 10 (T2) or 11 to 15 (T3) of an estrous cycle (five per treatment). Onset of estrus was designated as d 0 of the estrous cycle. Zearalenone was added to the daily ration and EB was administered via an intramuscular injection. Blood samples were collected every 10 min over a 4-h period on the first 2 d prior to onset of treatment; the first, third and fifth days of treatment; and the first two and the fifth day after the end of the treatment periods. Gilts receiving EB and Z during T2 and T3 had longer (P less than .05) inter-estrous intervals than C gilts. The range in inter-estrous intervals for Z and EB treatments was 28 to 74 and 27 to 63 d, respectively. Mean plasma progesterone concentrations were elevated (P less than .05) during T2 and T3 in EB and Z-treated gilts when compared with C females. Estradiol benzoate treatment during T2 and T3 reduced (P less than .05) mean plasma luteinizing hormone (LH) concentrations more than C or Z treatments. Mean plasma concentrations of 13, 14-dihydro-, 15-keto-prostaglandin F2 alpha (PGFM) during T3 were higher (P less than .05) in C and Z gilts on d 13 and 15 post-estrus when compared with EB gilts.(ABSTRACT TRUNCATED AT 250 WORDS)

The IgE-dependent release of a Hageman factor cleaving factor from human lung.

Passively sensitized human lung fragments were shown to release a protease by an IgE-dependent mechanism which can cleave human Hageman factor (Coagulation Factor XII). This enzyme, lung Hageman factor cleaving factor, was partially purified by ion exchange chromatography and gel filtration and was shown to be a serine protease with an apparent molecular weight of 12,000-13,000. This protease appears to be unrelated to any known activator of Hageman factor by molecular weight and inhibition profile and was shown to be distinct from an IgE-dependent prekallikrein activator, as well as the kininogenase activity defined as basophil kallikrein of anaphylaxis. Although it appears marginally capable of activating Hageman factor, it rapidly cleaves and inactivates the activated form so that the net effect is a loss of activatable Hageman factor. The result suggests that diminished levels of Hageman factor that may be seen associated with IgE-dependent reactions can be due to digestion and depletion rather than activation, and other criteria for activation of the contact system must be employed.