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Background Leaders of Missouri's State Targeted Response to the opioid crisis (STR) grant have prioritized increasing access to treatment medications for opioid use disorder (MOUD) through a “Medication First” approach. This conceptual framework prioritizes rapid, sustained, low-barrier access to MOUD for optimal impact on decreased illicit drug use and mortality. Medication First principles and practices were facilitated through state-level structural changes and disseminated to participating community treatment programs via a multi-pronged, multi-disciplinary approach. In the first nine months of STR, 14 state-contracted treatment agencies operating 38 sites used STR funding to implement the Medication First model. Methods We utilized state billing and service data to make comparisons before and during STR on the following outcomes: MOUD utilization, timely access to MOUD, amount of psychosocial services delivered, treatment retention at 1, 3, and 6 months, and monthly price of treatment. We conducted follow-up analyses examining differences across MOUD types (no medication, methadone, buprenorphine, oral naltrexone, mixed antagonist + agonist, and extended release naltrexone). Results During STR, MOUD utilization increased (44.8% to 85.3%), timeliness of MOUD receipt improved (Median of 8 days vs. 0 days), there were fewer psychosocial services delivered, treatment retention improved at one, three, and six month timeframes, and the median cost per month was 21% lower than in the year prior to STR. All differences were driven by increased utilization of buprenorphine. Conclusions Findings suggest Medication First implementation through STR was successful in all targeted domains. Though much more work is needed to further reduce logistical, financial, and cultural barriers to improved access to maintenance MOUD, the steps taken through Missouri's STR grant show significant promise at making swift and drastic transformations to a system of care in response to a growing public health emergency.

To identify antenatal and intrapartum risk factors for neonatal hypoxic ischemic encephalopathy (HIE). A single center, retrospective cohort study was conducted for 25,494 singleton births ≥36 weeks’ gestation born between 2009 and 2016. Univariate and multivariate analyses were performed to identify risk factors for HIE. Thirty-seven infants met HIE inclusion criteria. Independent antenatal risk factors included primigravida, previous fetal death/stillbirth, antidepressant use, illicit drug use, Rh sensitization, and adjusted gestational weight gain >13.6 kg. Independent intrapartum risk factors identified were placental abruption, ruptured uterus, moderate-to-heavy meconium stained amniotic fluid, and delivery by cesarean-section. An intrapartum risk factor was present in 70.3% of the HIE group compared with 29.6% of the non-HIE group. Intrapartum period risk factors appear to be important for the development of HIE. Gestational weight gain may serve as an important modifiable factor to reduce the risk of HIE.

Background The sharp rise in opioid use disorder (OUD) among women coupled with disproportionally high rates of unintended pregnancy have led to a four-fold increase in the number of pregnant women with OUD in the United States over the past decade. Supporting intentional family planning can have multiple health benefits and reduce harms related to OUD but requires a comprehensive understanding of women’s perspectives of preventing unintended pregnancies. The purpose of this study was to comprehensively evaluate the knowledge, attitudes and experiences as they relate to seeking contraception, particularly LARCs, among women with active or recovered opioid misuse. Methods In-depth interviews and focus group discussions with 36 women with current or past opioid misuse were recorded and transcribed. Transcripts were coded by ≥ 2 investigators. Themes related to contraceptive care seeking were identified and contextualized within the Health Belief Model. Results Our analysis revealed seven interwoven themes that describe individual level factors associated with contraceptive care seeking in women with current or past opioid misuse: relationship with drugs, reproductive experiences and self-perceptions, sexual partner dynamics, access, awareness of options, healthcare attitudes/experiences, and perceptions of contraception efficacy/ side effects. Overall, perceived susceptibility and severity to unintended pregnancy varied, but most women perceived high benefits of contraception, particularly LARC. However, perceived barriers were too high for most to obtain desired contraception to support family planning intentions. Conclusions The individual-level factors identified should inform the design of integrated services to promote patient-centered contraceptive counseling as a form of harm reduction. Interventions should reduce barriers to contraceptive access, particularly LARCs, and establish counseling strategies that use open, non-judgmental communication, acknowledge the continuum of reproductive needs, explore perceived susceptibility to pregnancy, and utilize peer educators.

Objectives Canine-assisted therapy (CAT) has been used in many settings with much success, yet no study has assessed its feasibility and receptiveness in hospitalized patients awaiting heart transplantation. Methods Patients admitted to our institution with a status I for heart transplantation during a 12-month period (April 2014-April 2015) were prospectively included in a feasibility pilot study. Patients were included if there was no history of transmittable disease or active infectious process and consented for study participation. Each patient was visited daily by a canine and quantitative and qualitative data regarding the visit were obtained. Results A total of 11 patients were included in the study. Most patients were men (n = 8, 72.7%) and the average age was 51.1 years. A total of 146 individual therapies took place, totaling 2718 minutes of CAT during the study period. Each patient had an average of 13.3 visits and each visit had an average duration of 14.7 minutes. Patient receptiveness, as measured by the CAT volunteer, averaged 9.9 (scale 0-10). No reports of infection transmission occurred. Conclusions Our study found that CAT among hospitalized pre-heart transplant patients is feasible and is a welcomed adjunct to usual medical care.

Elevated mean right atrial pressure (RAP) measured by cardiac catheterization is an independent risk factor for mortality. Prior studies have demonstrated a modest correlation with invasive and noninvasive echocardiographic RAP, but the prognostic impact of estimated right atrial pressure (eRAP) has not been previously evaluated in patients with pulmonary arterial hypertension (PAH).A retrospective analysis of 121 consecutive patients with PAH based on right-sided heart catheterization and echocardiography was performed. The eRAP was calculated by inferior vena cava diameter and collapse using 2005 and 2010 American Society of Echocardiography (ASE) definitions. Accuracy and correlation of eRAP to RAP was assessed. Kaplan-Meier survival analysis by eRAP, right atrial area, and Registry to Evaluate Early and Long-term PAH Disease Management (REVEAL Registry) risk criteria as well as univariate and multivariate analysis of echocardiographic findings was performed.Elevation of eRAP was associated with decreased survival time compared with lower eRAP (P.001, relative risk = 7.94 for eRAP15 mm Hg vs eRAP ≤ 5 mm Hg). Univariate analysis of echocardiographic parameters including eRAP15 mm Hg, right atrial area18 cm², presence of pericardial effusion, right ventricular fractional area change35%, and at least moderate tricuspid regurgitation was predictive of poor survival. However, multivariate analysis revealed that eRAP15 mm Hg was the only echocardiographic risk factor that was predictive of mortality (hazard ratio = 2.28, P = .037).Elevation of eRAP by echocardiography at baseline assessment was strongly associated with increased risk of death or transplant in patients with PAH. This measurement may represent an important prognostic component in the comprehensive echocardiographic evaluation of PAH.

A 48-year-old woman presented to our emergency department with progressive dyspnea, cough with clear sputum, and a 9-kg weight loss. Evaluation at an outside emergency department 6 months previously had revealed a right middle lobe infiltrate on chest radiography (CXR), and she was treated with a 10-day course of levofloxacin for presumed community-acquired pneumonia. Her symptoms continued, and 1 month later, she went to an outside pulmonologist who found bilateral alveolar infiltrates on chest computed tomography (CT).

Gonadotropin-releasing hormone (GnRH) has been shown to play a role in the regulation of human chorionic gonadotropin (hCG) secretion by the human placenta. Molecular studies have demonstrated that human placental trophoblast cells synthesize a progonadotropin-releasing hormone (pro-GnRH) identical to its human hypothalamic counterpart. However, far less is known about nonhuman primates. To determine whether pro-GnRH exists in the rhesus placenta, pro-GnRH mRNA was cloned, sequenced, and shown to be 97.6% homologous to its human placental counterpart. A single base difference (base 1167) in the domain encoding GnRH results in the same amino acid, arginine, in position 8, whereas four base differences (bases 1200, 1253, 1268, 1292) in the domain encoding GnRH-associated peptide (GAP) result in four different amino acids in positions 19, 37, 42, and 50. The absence of a basic amino acid in position 50 suggests the rhesus sequence may be cleaved to yield GAP peptides different from the human placenta. Thus, these data justify the use of mammalian GnRH in studies of rhesus placental function, but indicate the need to investigate the roles unique GAP peptides may play in placental/uterine function.

Studies to date have demonstrated epidermal growth factor (EGF) receptors primarily on the outer plasma membrane of the human placental syncytiotrophoblasts facing maternal blood and to a lesser extent on the cytotrophoblast stem cells. In the present studies, first- and third-trimester human placental tissues were immunostained with monoclonal antibodies (MAb) to the EGF binding domain of the human EGF receptor or to the activated (tyrosine-phosphorylated) human EGF receptor. Cytotrophoblasts, syncytiotrophoblasts, and fetal connective tissue cells in first-trimester tissues immunostained with both MAb, with the notable exception of the absence of staining of activated EGF receptor over cytotrophoblast plasma membranes. In contrast, staining of third-trimester placentas with either MAb yielded little to no staining of either trophoblast cell layer but intense staining of fetal connective tissue cells. Staining for EGF receptors over cytotrophoblasts in the first trimester is consistent with the hypothesis that maternal EGF or TGF-alpha derived from the endometrium or placenta may be the mitogen responsible for cytotrophoblast cell division and that the receptors localized to the syncytiotrophoblast are involved in EGF regulation of differentiated function. The absence of heavy staining of activated EGF receptor on trophoblast plasma membranes in third-trimester placentas is consistent with down-regulation of EGF receptor activity.

GnRH has been shown to play a role in the regulation of secretion of hCG by human placenta. Immunocytochemical studies have demonstrated the presence of the peptide, but do not address whether the GnRH is maternal, fetal, or placental in origin. In situ hybridization studies using a biotinylated pro-GnRH cDNA were, therefore, undertaken to determine the distribution of pro-GnRH mRNA in first trimester placental samples. Using an avidin-biotin-Cy.5 detection system in conjunction with laser scanning confocal microscopy, pro-GnRH mRNA was shown to be present in both the cytotrophoblast and syncytiotrophoblast cells of placental villi, although not in the cells of the fetal connective tissue. Prior hybridization with a 200-fold excess of unlabeled probe blocked hybridization of the labeled pro-GnRH probe. Southern and sequence analysis demonstrated that the probe hybridized to a transcript identical to hypothalamic GnRH. Immunocytochemical staining using an antiserum to amino acids 6-16 of pro-GnRH demonstrated the presence of translated pro-GnRH in both the cytotrophoblast and syncytiotrophoblast epithelia. We conclude that the synthesis of pro-GnRH by both the cytotrophoblast and the syncytiotrophoblast of human placenta is consistent with either an autocrine or a paracrine mode of GnRH regulation of hCG secretion.

Although African American women have an overall lower incidence of breast cancer, African American women40 years of age are more likely than Caucasian women of all ages and postmenopausal African American women to be diagnosed with breast cancer and exhibit tumor characteristics associated with poorer survival. To begin to address this disparity, studies must be conducted to examine breast cancer preventive factors in this subpopulation of women. However, the strategies needed to recruit younger African American women have not been well defined.In this study, we assessed methods used for recruiting and retaining healthy premenopausal African American women into the African American Nutrition for Life (A NULIFE) Study. The number of women contacted, enrolled, and retained by each recruitment strategy and the efficiency of individual strategies were calculated.Overall, recruitment through social networking was most effective in contacting large numbers of healthy premenopausal African American women. The worksite recruitment method was the most efficient recruitment strategy employed, with a ratio of 40%. The study participants (n = 164) were more likely to beor=35 years of age and have completed some college. Additionally, the interpersonal relationships recruitment approach proved most efficient (33%) in retaining participants who completed the yearlong study.The findings from this study add to the evolving research literature on minority recruitment strategies for research studies but specifically address effective recruitment of healthy young premenopausal African American women. The results demonstrate the need to use multiple recruitment strategies when recruiting this subgroup of African American women.

An immunocytochemical assay for estrogen receptor (ER) was used to study the distribution of receptor in fetal and immature female mouse reproductive tracts. Immunoblots confirmed that a single band, the size of the ER, immunostained in extracts from day 15 and 17 fetal reproductive tracts. Staining was observed over nuclei of epithelial cells of the Mullerian duct and over nuclei of cells of the developing connective tissue (mesenchymal cells) of the reproductive tract on fetal day 15. By day 17 when a primitive uterus could be distinguished, ERs were detected in nuclei of mesenchymal cells, but in only a small portion of epithelial cells. A different pattern of immunocytochemical staining was observed in uteri from animals killed on the day of birth; cells of the connective tissue contained ER, but the epithelial cells did not. By 4 or 6 days after birth, more nuclei in the connective tissue stained for ER with a greater intensity compared to nuclear staining in epithelial cells. ERs were detectable in nuclei of both uterine epithelial cells and connective tissue cells on days 10 and 19 after birth.

To execute interdisciplinary care for complex dentofacial abnormalities, a team of providers must develop a consistent philosophy and mission regarding examination, diagnostic, and treatment planning procedures. To obtain optimum therapeutic results, patients must rely on the collaborative expertise of a team. This article and case study illustrates a multidisciplinary team's role in the prosthetic and esthetic rehabilitation of the maxillary anterior region.

Gonadotropin-releasing hormone (GnRH) interacts with a putative receptor in human placenta to cause the dose-dependent release of human chorionic gonadotropin (hCG) in a manner analogous to hypothalamic GnRH stimulation of luteinizing hormone (LH), and follicle-stimulating hormone (FSH) by the pituitary gland. However, GnRH agonists bind a placental binding site at a lower affinity than they bind the pituitary GnRH receptor, suggesting the two sites differ. To address this issue, human placental GnRH receptor mRNA from an 8-wk placental sample was amplified using primers based on the sequence of the human pituitary receptor, cloned, and sequenced. The sequence of the placental transcript was found to be identical to its pituitary counterpart. Thus, as with the pituitary receptor, this placental receptor transcript appears to encode a single polypeptide chain with seven intramembranous domains and two glycosylation sites. However, it is possible these receptors differ in their posttranslational processing or there is more than one placental GnRH receptor. In addition, subsequent gene amplification studies, modified to increase the sensitivity of the method, revealed a band of the predicted size (approximately 1051 bp) in a 6-wk and two 8-wk placental samples, but not in 5-wk and 7-wk samples. The failure to amplify GnRH receptor mRNA at these gestational ages did not appear to be an issue of cell health or sample degradation, since GnRH, alpha hCG, and beta hCG mRNA were amplified from each of these tissues. Instead these data suggest GnRH receptor mRNA may have a short half-life or the mRNA may be translated as rapidly as it is transcribed, with the result that it has not accumulated in the cell.

A primary cell culture system was developed to study the epidermal growth factor (EGF) receptor in the fetal bovine mesonephros and its urogenital derivatives. Radioreceptor assays demonstrated EGF binding as early as Day 37 in mesonephric cells and in cells derived from the fetal reproductive ducts, gonads, and metanephros--all mesonephric derivatives. Immunocytochemical studies revealed that EGF receptors were localized in the ductal and tubular epithelium of these urogenital organs. EGF induced DNA synthesis and tyrosine phosphorylation in the bovine mesonephric cells, suggesting that EGF receptors detected in these cells were functional. In addition, transforming growth factor alpha, the putative fetal ligand for the EGF receptor, was found to specifically compete for EGF binding to the receptor, as well as to induce DNA synthesis in a manner similar to that of EGF. Estrogen did not regulate EGF receptors or specifically bind to bovine mesonephric cells. The development of estrogen receptors in the bovine species occurs markedly later than that of the EGF receptor, in contrast to the mouse, where both receptors are observed at very early stages of reproductive tract development.

To date, there is no conclusive evidence that ERs are present in preimplantation embryos. There are reports that estrogen is made by the rabbit blastocyst (61), and estrogens have been used to induce implantation in mice (62), but whether estrogens act through ERs in the embryo or in the maternal uterus is not known. ERs may be present in early embryos, but if so, levels are below the methods of detection used thus far. Perhaps with more sensitive immunodetection methods, it may be possible to detect ERs in embryos if they are present. Using PCR, messenger RNA for ER has been detected as early as the oocyte stage in mouse embryos (Q. Hou and J. Gorski, unpublished results). This was confirmed recently by Wu et al. (83a). Figure 7 shows a model for the pattern of ER expression in the developing mouse fetus based on the various reports discussed in this review. ERs are present in the 10-day mouse fetus, possibly in the developing ambisexual reproductive tract. Analysis of seven individual 10-day-old fetuses taken from the same litter showed similar levels of an immunostained protein the size of the ER in each fetus (57). The pattern of expression of ER between implantation and the development of the reproductive tract may be the same in male and female mice. Estrogen, acting through ERs, may be one factor (of many) that determines which cells are destined to be part of the indifferent reproductive tract. We were not able to isolate fetal mouse reproductive tracts at an indifferent stage (day 10) due to their very small size. One way to study ER in the indifferent reproductive tract would be to examine these tissues in a larger animal, such as the bovine, using similar immunodetection methods. The distribution of ER in the fetal mouse reproductive tract on fetal days 13 (before sexual differentiation) and 15 (initiation of sexual differentiation) is similar in males and females (71, 72). Thus, estrogen does not appear to be responsible for the initiation of sexual differentiation. Early experiments by Jost (41) showed that removal of the gonad from male or female rabbit fetuses resulted in the female phenotype, which lent weight to the hypothesis that ovarian hormones are not critical in the development of the female phenotype, whereas testicular hormones are essential for the development of the male phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunodetection methods were used to detect estrogen receptors (ER) in male reproductive tracts on fetal days 13, 15, and 17 and on the day of birth. Immunocytochemistry revealed that most of the cells of the gonad and associated Wolffian duct stained for ER on fetal day 13. During the next 6 days, ER distribution changed, and by the day of birth, ERs were observed only in epithelial cells of the epididymis (derived from the Wolffian duct) and in a portion of cells from the testis. Immunoblots confirmed that a band the size of the ER stained in reproductive tracts for all ages studied. Similar to the fetal female, ERs are present throughout the early development of the fetal male reproductive tract. However, in contrast to the female, ERs appear to decrease in the male fetal reproductive tract at the time of birth.

Hormones related to the pituitary hormones PRL and GH are produced by the utero-placental unit of many species. In the cow, these include bovine placental lactogen (bPL) and a distantly related subfamily including the protein encoded by bovine PRL-related complementary DNA I (bPRCI). In the present studies, we defined the onset of expression of these genes in order to begin to study the regulation of their expression and function before implantation. Messenger RNA levels of both bPL and bPRCI were assessed by dot blot analysis of total RNAs prepared from conceptuses on days 15-25. Specific transcripts were not detectable at day 15 but were readily apparent beginning at day 17. To define the portions of the placenta responsible for expressing these genes, RNA was prepared from chorion (separated into cotyledons and intercotyledonary membrane), allantois, and amnion from day 58 gestation and analyzed by Northern hybridization. Transcripts for both these hormones were confined to the chorion, with intercotyledonary RNA containing at least as much as that prepared from cotyledons. To localize the product of these genes, extraembryonic membranes were immunostained with bPL and bPRCI antisera. Mononucleated trophoblastic cells stained for bPL and bPRCI at day 18; similar staining was apparent at days 23 and 24, as well as in some but not all binucleated cells. Microsomes prepared from endometrium exhibited specific binding of bPL throughout pregnancy as well as during the luteal phase of the cycle. At midgestation, these high affinity, low capacity receptors (dissociation constant of 1.27 x 10(-10) M, maximum binding capacity of 44.2 fmol/mg protein) demonstrated a selective affinity for bPL in preference to bGH and bPRL. Using this radioreceptor assay, we demonstrated the presence of bPL at nanomolar concentrations in washings from the uterine lumen, suggesting that bPL may exert paracrine effects on the endometrium.

Two patients with enlarged pituitary fossae presented with a number of features in their medical history and laboratory workup suggesting the diagnosis of “non-functioning” pituitary adenoma. Both patients had low serum thyroxine (T4) concentrations and resin T3 uptake, and one patient showed a high titer of microsomal thyroid antibodies. Both patients had high serum TSH concentrations before and after stimulation with TRH. After pituitary surgery, the serum TSH levels diminished but remained abnormally high and were suppressed by thyroid hormone administration. Light microscopic studies of the tumors, including differential staining and immunocytochemical procedures for the demonstration of TSH cells, showed cells indistinguishable from those of chromophobic adenomas. Ultrastructurally, the adenoma cells contained relatively few granules, similar to those described in “non-functioning” chromophobic tumors. Our investigations suggest that some of the patients diagnosed as having “non-functioning” pituitar...

The bovine fetal placenta expresses a family of PRL-related genes, consisting of the gene encoding bovine placental lactogen (bPL), and a diverse group of related genes, exemplified by bovine PRL-related cDNA I (bPRCI). bPL and the protein encoded by bPRCI are quite distinct from one another, predicting proteins only about 36% similar in amino acid sequence. To identify the cells responsible for the expression of bPL and bPRCI, in situ hybridization experiments were performed. 35S-Labeled RNA probes were prepared from the 3' regions of bPL and bPRCI and allowed to hybridize to frozen sections of bovine placentomes. Transcripts corresponding to bPL and bPRCI colocalized to fetal binucleate cells, which is consistent with the immunocytochemical localization of bPL. Signal from radiolabeled antisense strand probe was blocked by pretreatment of the sections with a 150-fold excess of unlabeled probe, but not by an excess of unlabeled probe prepared using the other cDNA as a template. This demonstrated that the signals observed were specific for bPL and bPRCI and that the two probes did not cross-hybridize at the stringency of our conditions. Radiolabeled sense strand probes yielded no signal. We conclude that the binucleate cells of the bovine placenta transcribe at least two members of the PRL gene family that may influence fetal development and maternal adaptation to pregnancy.

A pituitary adenoma with suprasellar extension that had caused hyperthyroidism due to secretion of excess thyrotropin (TSH), as well as mild hyperprolactinemia, was studied with differential staining, immunocytochemistry and electron microscopy. Most cells of the tumor strained lightly with aldehyde thionin, which demonstrates the granules of normal thyrotrops, and immunocytochemically with antiserum to the hormone-specific beta chain of TSH. A minority of the cells was immunoreactive for prolactin. Electron microscopy revealed light cells interspersed with highly pleomorphic dark cells. Both were sometimes multinucleated, and contained variable numbers of small secretion granules, multiple Golgi complexes, and abundant endoplasmic reticulum.

42 women with amenorrhœa and hyperprolactinaemia had trans-sphenoidal surgery and resection of histologically verified pituitary adenomas. 74% of these patients developed amenorrhœa and/or galactorrhœa in immediate association with the use or discontinuation of oral contraceptives or post partum. There was enough adenomatous tissue for immunocytochemical studies in 35 specimens and specific localisation of prolactin was possible in 31. There is evidence that about 10% of the population have small pituitary tumours, and the majority of these tumours, though asymptomatic, are potentially prolactin-secreting. It is suggested that œstrogens, which are known to modulate prolactin secretion in normal human beings and in animals, can induce the growth and expression of otherwise silent pituitary lesions and that this should be considered a risk of oral-contraceptive use.

Bovine placental lactogen (bPL) has been isolated from bovine trophoblast and characterized as a 32 K mol wt protein which exists in three different forms which differ in their isoelectric point values and their amino acid compositions. Two of the three forms have been shown to have both bovine GH (bGH)- and bovine PRL (bPRL)-like activities equal on a molar basis to bGH and BPRL in radioreceptor assays. It has been postulated that, in sheep, PL is delivered to the maternal circulation by the migration of fetal binucleate cells from the trophoblast across the fetal-maternal boundary into the uterine epithelium. To determine whether an analogous situation exists in the cow, antibodies to bPL were used to localize bPL in bovine placentomes and to measure its concentration in fetal and maternal sera. For cytology, bPL was localized on sections of placentomes from midgestation and term bovine placentas using an indirect immunoperoxidase technique. Stained binucleate cells were demonstrated throughout the trophoblast, often in close association with the microvillous boundary which separates the trophoblast from the maternal epithelium. In cross-sections of fetal villi, binucleate cells with cytoplasmic processes extending into and through the uterine epithelium were immunostained as well as cells within the plane of the uterine epithelium in close approximation or apposition to the maternal basement membrane. RIA demonstrated bPL to be present in maternal sera in concentrations of 1-2 ng/ml and in fetal sera at 5-12 ng/ml. These data are consistent with the hypothesis that binucleate cell migration accomplishes the delivery of bPL to the maternal circulation.

Growth hormone and prolactin cells were immunostained in human hypophyses with antibody against rat growth hormone or prolactin and the peroxidase-antiperoxidase complex. Growth hormone cells were round and, in normal pituitaries, arranged in sizable groups. Prolactin cells occurred singly and were less numerous; they were often extensively branched. Only a few prolactin cells stained with carmoisine. Incubation of the antibody with an excess of the appropriate antigen greatly diminished or abolished immunostaining; absorption of anti-prolactin with growth hormone often enhanced it. Prolactin cells were somewhat hypertrophied and hyperplastic in a neonate. Many of them stained with carmoisine. An even greater hypertrophy and hyperplasia of these cells (which pushed apart the growth hormone cells) was found in a lactating woman. Immunostained giant prolactin cells were also observed. Staining of the prolactin cells with carmoisine was extensive. Upon prolonged exposure to anti-growth hormone antibody, ACTH/MSH cells also showed immunostaining which was abolished by absorption of the antiserum with growth hormone but not with synthetic 1-24ACTH. Growth hormone cells evidently correspond to the alpha acidophils of Romeis, prolactin cells in lactation to his eta cells; the relation of his epsilon cells to the pleomorphic "resting" prolactin cells is not clear.

Previous work has shown that, unlike other species, placental lactogen (PL) in the bovine (bPL) has a mol wt of approximately 32,000 and exists in several different forms with different isoelectric points. This study was carried out to develop a more rapid purification scheme, whereby the yield of bPL obtained was increased while at the same time the possibility of artifacts from a prolonged purification protocol was decreased. A procedure was developed in which a fraction enriched in bPL-containing granules was obtained after gentle disruption of the binucleate cells of the fetal cotyledon. The fetal portion of the placentomes from midpregnant cows was minced with scissors and vigorously stirred in order to remove and disrupt binucleate cells within the fetal villi. The supernatant from this step was fractionated by differential centrifugation followed by a four-step discontinuous Percoll gradient of 1.03-1.08 g/ml. A granule-enriched fraction was isolated from the 1.04 g/ml zone from which membrane-enclosed protein was released by freezing and thawing. Membranes and insoluble proteins were sedimented by high speed centrifugation to yield an extract which contained approximately 20% of the hormone initially in the tissue. Two subsequent chromatographic steps, gel filtration on Sephadex G-75 and high performance reversed phase chromatography with a C-4 column, resulted in a preparation of greater than 98% homogeneity. Two-dimensional gel electrophoresis of purified bPL revealed at least nine protein spots in the 31,000-33,000 mol wt range with isoelectric points ranging from 4.85-6.3. All forms exhibited parallel dilution curves in a RIA for bPL. It would appear, therefore, that multiple forms of bPL exist and that they are not artifacts of the prolonged purification protocol previously used.

JB4 and Immunobed are water-soluble embedding media used for embedding large blocks of tissue. Immunobed was specifically designed for immunocytochemistry because ethanol extraction of an additive in the monomer of the resin is reported to render tissue sections permeable to immunoglobulins. We have modified the manufacturer's protocol to accomplish localization of two protein antigens in tissues embedded in either JB4 or Immunobed. Luteinizing hormone-beta (LH beta) was localized in sections of rat and bovine pituitary tissues and bovine placental lactogen (bPL) was localized in sections of placentomes from bovine placentas. Sections received one of the following pre-treatments: absolute EtOH; NaHCO3 buffer, pH 6-10; EtOH followed by NaHCO3 buffer; one of several enzymes; EtOH followed by enzyme; NaHCO3 buffer followed by enzyme. Anti-LH beta stained only pituitary gonadotrophs and anti-bPL stained only placental binucleate cells, as assessed by absorption controls. Pre-treatment with enzyme was required for staining of sections, but an alkaline pH change (NaHCO3) had little or no effect. Ethanol pretreatment had little or no effect alone or in conjunction with NaHCO3 or enzyme. Sections were sufficiently thin (1.5 micron) to afford resolution of structure, but suitably large (approximately 2 cm2) to minimize problems of sampling.

Among 92 surgically removed pituitary adenomas immunostained for prolactin and growth hormone, 70 showed positive staining for prolactin. The majority of these (54) was associated with hyperprolactinemia leading to amenorrhea (and often galactorrhea) in women of reproductive age. Similar tumors, asymptomatic or conducive to disturbances of sexual function, were found in six hyperprolactinemic men. Among nine acromegalics, seven had immunostained lactotrophs associated with the somatotrophic adenomas cells, but only two of these had hyperprolactinemia. In all of the remaining tumors that had at least some immunoreactive lactotrophs, mild hyperprolactinemia had been present This indicates that immunostaining of pituitary tumors for prolactin correlated well with elevated plasma prolactin levels, except in the case of mixed somatolactotrophic adenomas. The patterns of distribution of immunoreactive prolactin in adenoma cells are illustrated. Since only some of the prolactin-producing adenomas stained with carmoisine--a dye that has been suggested as a marker for prolactin cells--immunocytochemistry is the method of choice for the identification of prolactin-secreting adenomas.

Immunocytochemical staining using the unlabeled antibody peroxidase-antiperoxidase method was undertaken to localize and characterize in ultrathin sections of human pituitaries the cells responsible for the secretion of GH and PRL. Somatotrophs in seven pituitaries stained with human (h) PRL-absorbed antiserum to hGH, were abundant, round to ovoid, densely granulated cells, whose mean (+/-SD) granule diameter was 368 +/- 60 nm. Lactotrophs immunostained with antiserum to hPRL were less numerous, angular or branching cells, with fewer round to ovoid granules, the mean diameter (+/-SD) of which was 185 +/- 35 nm in six pituitaries. The somewhat larger PRL granules (up to a mean diameter of 360 nm) seen in two of three additional pituitaries may have been related to the previous therapeutic administration of estrogen. Whereas the immunostained GH-secreting cells resemble the presumed somatotrophs identified in other studies on the basis of nonimmunological staining, the immunostained PRL-secreting cells differ considerably from the cells with large (600--1000 nm) granules designated as lactotrophs by several previous investigators. The hazards of ultrastructural identification of human pituitary cell types on purely morphological (as opposed to immunocytochemical) grounds are emphasized.