An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis.

An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent.     

Effects of food deprivation on cocaine base smoking in rhesus monkeys

Studies have shown that both food deprivation and response cost have important influences on the magnitude of self-administration of a wide variety of psychoactive drugs. In an attempt to extend these findings to the smoked route of drug self-administration, the effects of food allotment and fixed-ratio (FR) value were evaluated in four male rhesus monkeys trained to smoke cocaine base. In the first phase of the experiment, monkeys were trained to self-administer smoked cocaine base under a chained progressive-ratio (PR), fixed-ratio (FR) schedule during daily experimental sessions. Monkeys were required to make 20 lever-press responses and then five inhalations on a smoking spout to obtain the first smoke delivery. The lever ratio then increased to 60, 140, 300, 620, 1260, 2540, and 4940 for each successive smoke delivery. The initial lever ratio value was reset to 20 at the beginning of each daily session. The body weights of three monkeys were determined under free-feeding conditions. Monkeys were then restricted to 100 g food and, when body weights had stabilized, the daily food allotment was increased to 150 g, approximately 210 g, or greater than 400 g (satiation). As the daily food allotment and body weight increased, the mean number of smoke deliveries decreased in two of three monkeys. In the second phase of the experiment, three monkeys were maintained under either food-satiated or food-restricted conditions. Body weights were maintained at approximately 90% of their free-feeding weights under food-restricted conditions. The cost of the drug (lever FR value) was constant within each experimental session, but was increased after 3 consecutive days of stable responding. Fixed-ratio values were increased from 128 to 256, 512, 1024, and 2048. Monkeys were required to complete the lever FR value and then to make five inhalations on the smoking spout to gain access to 1.0 mg/kg per delivery cocaine base. The mean number of smoke deliveries increased at FR 256, 512, and 1024 when monkeys were food-restricted as opposed to food-satiated. Correspondingly, the mean number of responses increased under food-restricted conditions. Responding continued to increase over a wider range of FR values, and the peak number of responses was higher under food-restricted, as opposed to food-satiated conditions. These results, using the smoking route of administration, are consistent with the hypothesis that food deprivation increases the self-administration of reinforcing drugs.     

Immunological characterization of dehydroepiandrosterone sulfotransferase from human liver and adrenal.

Dehydroepiandrosterone sulfotransferase (DHEA-ST), a steroid sulfotransferase (ST), has recently been purified from human liver cytosol and partially characterized. DHEA-ST has a subunit molecular mass of 35 kDa and is responsible for the majority of the sulfation of steroids and bile acids in the liver. For these studies, polyclonal antibodies to human liver DHEA-ST were raised in rabbits. The anti-human liver DHEA-ST antibodies were used to characterize the immunoreactivity of DHEA-ST in human liver and to study the relationship of human adrenal DHEA-ST to the liver form of the enzyme. Immunoblot analysis of several different human liver cytosol samples with the rabbit anti-human liver DHEA-ST antiserum detected only a single 35-kDa protein in each liver. Anti-human liver DHEA-ST antibodies also did not react with either form of phenol sulfotransferase (PST), P-PST or M-PST, present in human liver cytosol. DHEA-ST activity was purified from the 100,000 x g supernatant fraction of human adrenal tissue by DEAE-Sepharose CL-6B chromatography and 3',5'-diphosphoadenosine-agarose affinity chromatography. Human adrenal DHEA-ST was shown to have a molecular mass of 35 kDa, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of human adrenal cytosol revealed that the anti-human liver DHEA-ST antibodies reacted specifically with the 35-kDa subunit of DHEA-ST. The apparent Km values for DHEA and 3'-phosphodenosine-5'-phosphosulfate obtained with human adrenal DHEA-ST were 1.0 microM and 1.6 microM, respectively. Adrenal DHEA-ST demonstrated the same pattern of reactivity towards different steroid substrates as did human liver DHEA-ST, and neither form of DHEA-ST was found to sulfate cortisol. The results of this study suggest that DHEA-ST is the major steroid ST present in human liver and adrenal tissue and that the physical, biochemical, and kinetic properties of adrenal DHEA-ST are similar if not identical to those of the liver form of the enzyme.     

Escape responses following elimination of the giant interneuron pathway in the cockroach, Periplaneta americana

The entire set of giant interneurons (GIs) in the nerve cord of the cockroach, Periplaneta americana, was ablated using either electrolytic or surgical techniques. All animals with these lesions were capable of turning and running away from standard wind puffs. However, all animals responded much less frequently to standard wind stimuli following lesion, and the latency of their responses was significantly increased. These results are discussed in terms of a GI role in extremely short latency escape responses, and the idea that non-GI pathways, perhaps associated with head sensory structures, need to be considered in the normal control of escape in the cockroach.     

Application of a marker of ciliated epithelial cells to gynaecological pathology

BACKGROUND: The assessment of neoplastic disease in gynaecological histopathology can be complicated by the high incidence of metaplasia seen in tissues of the female genital tract. There is a need to identify specific tissue markers which can be applied in routine histopathological practice. AIM: To examine the clinical potential of a monoclonal antibody, LhS28, which reacts with basal bodies of ciliated epithelial cells. METHODS: A panel of normal and pathological gynaecological tissues was processed and labelled with LhS28. RESULTS: LhS28 immunoreactivity was found in the normal Fallopian tube where it was confined to ciliated rather than secretory epithelial cells. In the remaining specimens, LhS28 was associated exclusively with ciliated cells in tubal metaplasias of the cervix and endometrium and in benign serous lined inclusion cysts. CONCLUSIONS: LhS28 may be a valuable marker for identifying metaplasia of tubal type and may find application in distinguishing tubal metaplasia from low grade cervical glandular intraepithelial neoplasia.     

Induction of a differentiated ciliated cell phenotype in primary cultures of Fallopian tube epithelium

Human Fallopian tubal epithelial cells in culture lose morphological features associated with the epithelium in situ and the extent to which they retain their in-vivo phenotype or function is unknown. In order to address this question, immunocytochemical markers were identified which distinguish secretory (HMFG2+, LhS28-) from ciliated (HMFG2-, LhS28+) epithelial cells in tissue sections of Fallopian tube. These markers were used to analyse the phenotype of tubal cells in vitro. Primary cultures of human tubal epithelial cells were seeded onto glass and grown to confluence before addition of oestradiol-17beta. In the absence of hormone, tubal epithelial cells expressed cytokeratins and nuclear receptors for oestrogen and progesterone and adopted a homogeneous (HMFG2+, LhS28-) secretory cell phenotype. Following the addition of oestradiol-17beta, a proportion of cells became positive for LhS28. The induction of a ciliated epithelial cell phenotype was confirmed by scanning electron microscopy, where on permeable collagen membranes, approximately one-third of tubal epithelial cells became ciliated in the presence of oestradiol-17beta. We suggest that in vitro, tubal epithelial cells adopt an immature secretory-like phenotype and that oestrogen can induce differentiation to a ciliated epithelial cell phenotype.      

Local bioactive tumour necrosis factor (TNF) in corneal allotransplantation

The aim of this study was to examine the kinetic profile of bioactive TNF levels in aqueous humour of rabbit eyes undergoing corneal allograft rejection and to investigate the effect of locally blocking TNF activity after corneal transplantation. In a rabbit corneal transplantation, endothelial allograft rejection was identified and correlated with increase in central graft thickness. Samples of aqueous humour obtained on alternate days following transplantation were tested for TNF mRNA and bioactive TNF protein. To investigate the effect of locally blocking TNF activity in allograft recipients, the fusion protein TNFR-Ig was administered by injections into the anterior chamber after transplantation. Pulsatile increases in levels of this cytokine were found in 14 of 15 allograft recipients. Peaks of TNF bioactivity preceded by varying intervals the observed onset of rejection in allograft recipients. TNF levels were not elevated in aqueous humour from corneal autograft recipient controls or in serum of allografted animals. mRNA levels were elevated before onset of and during clinically observed allograft rejection. In three of seven animals receiving TNFR-Ig injections on alternate days from day 8 to day 16 post-transplant, clear prolongation of corneal allograft survival was demonstrated. Bioactive TNF is present in aqueous humour following rabbit corneal allotransplantation. Rather than correlating directly with endothelial rejection onset, pulsatile peak levels of TNF precede and follow the observed onset of endothelial rejection. Blockade of TNF activity prolongs corneal allograft survival in some animals, indicating that this cytokine may be a suitable target in local therapy of corneal allograft rejection.     

Advancing equity,diversity, and inclusion in the American College of Neuropsychopharmacology (ACNP): advances,challenges, and opportunities to accelerate progress

It is increasingly accepted that higher levels of excellence and innovation in research can be achieved by organizations that promote equity, diversity, and inclusion across several domains including ethnicity and gender. The purpose of this commentary is to provide an overview of the methods used to increase diversity within ACNP, as well as recommendations for accelerating progress. Annual membership surveys confirm increases in female membership and leadership positions, slower but encouraging signals for “Asian” and “Hispanic” members, and less progress for African American and other ethnic populations. Meetings have become visibly more diverse, due in part to ethnic minority travel awards and apparently increasing diversity among guest attendees. Evidence of increasing inclusion includes well-attended networking events and minority-relevant programming, active communications about diversity-related events and resources, and strong statements by ACNP leadership that embrace diversity as a core value and support collaboration among key committees and task forces to identify and implement pro-inclusion and diversity-enhancing efforts. We believe ACNP can accelerate progress with more scientifically valid approaches to assessing diversity and inclusion. The current membership survey includes five outmoded ethnic options and postmeeting surveys that are not designed to assess inclusion efforts and consequences. Measures should be developed that better characterize diversity and assess efforts to reduce the barriers that exist for potential non-White populations (e.g., annual membership and meeting attendance costs). Increased collaboration with NIH and other organizations that are committed to these same goals may also contribute to acceleration of progress by ACNP and other scientific organizations.Subject terms: Medical research, Neuroscience     

Identification and characterisation of ten microsatellite loci in the Noisy Scrub-bird Atrichornis clamosus using next-generation sequencing technology

The Noisy Scrub-bird is an endangered species of songbird endemic to the south coast of Western Australia that has undergone a major and prolonged population bottleneck. Using shotgun 454 next-generation DNA sequencing we have identified and characterised ten polymorphic microsatellite loci in this species. Observed allelic diversity was relatively low (2–5 alleles per locus) and significant deviations from Hardy–weinberg Equilibrium observed, although the presence of null alleles was only postulated for two loci. The microsatellite loci characterised in this study will be useful in a future population genetics studies in this endangered species.