Standards of Care for the Health of Transgender and Gender Diverse People, Version 8.

Background: Transgender healthcare is a rapidly evolving interdisciplinary field. In the last decade, there has been an unprecedented increase in the number and visibility of transgender and gender diverse (TGD) people seeking support and gender-affirming medical treatment in parallel with a significant rise in the scientific literature in this area. The World Professional Association for Transgender Health (WPATH) is an international, multidisciplinary, professional association whose mission is to promote evidence-based care, education, research, public policy, and respect in transgender health. One of the main functions of WPATH is to promote the highest standards of health care for TGD people through the Standards of Care (SOC). The SOC was initially developed in 1979 and the last version (SOC-7) was published in 2012. In view of the increasing scientific evidence, WPATH commissioned a new version of the Standards of Care, the SOC-8. Aim: The overall goal of SOC-8 is to provide health care professionals (HCPs) with clinical guidance to assist TGD people in accessing safe and effective pathways to achieving lasting personal comfort with their gendered selves with the aim of optimizing their overall physical health, psychological well-being, and self-fulfillment. Methods: The SOC-8 is based on the best available science and expert professional consensus in transgender health. International professionals and stakeholders were selected to serve on the SOC-8 committee. Recommendation statements were developed based on data derived from independent systematic literature reviews, where available, background reviews and expert opinions. Grading of recommendations was based on the available evidence supporting interventions, a discussion of risks and harms, as well as the feasibility and acceptability within different contexts and country settings. Results: A total of 18 chapters were developed as part of the SOC-8. They contain recommendations for health care professionals who provide care and treatment for TGD people. Each of the recommendations is followed by explanatory text with relevant references. General areas related to transgender health are covered in the chapters Terminology, Global Applicability, Population Estimates, and Education. The chapters developed for the diverse population of TGD people include Assessment of Adults, Adolescents, Children, Nonbinary, Eunuchs, and Intersex Individuals, and people living in Institutional Environments. Finally, the chapters related to gender-affirming treatment are Hormone Therapy, Surgery and Postoperative Care, Voice and Communication, Primary Care, Reproductive Health, Sexual Health, and Mental Health. Conclusions: The SOC-8 guidelines are intended to be flexible to meet the diverse health care needs of TGD people globally. While adaptable, they offer standards for promoting optimal health care and guidance for the treatment of people experiencing gender incongruence. As in all previous versions of the SOC, the criteria set forth in this document for gender-affirming medical interventions are clinical guidelines; individual health care professionals and programs may modify these in consultation with the TGD person.

Virally infected hepatocytes are resistant to perforin-dependent CTL effector mechanisms.

Cell-mediated cytotoxicity plays an important role in the clearance of noncytopathic viruses from infected tissues. Perforin-dependent cytotoxic mechanisms have been noted to play an important role in the clearance of infections from multiple extrahepatic organs. In contrast, mice with defects in the Fas/Fas ligand (FasL)-mediated cytotoxicity pathway exhibit delayed clearance of adenovirus from the liver without apparent delay in the clearance of viral infections from extrahepatic organs. The present studies examined the role of cytotoxic effector mechanisms in intrahepatic immune responses to a replication-defective, recombinant beta-galactosidase-encoding adenovirus (AdCMV-lacZ). Delayed clearance of AdCMV-lacZ from the livers of FasL-defective B6.gld mice, but not perforin-deficient B6.pfp(-/-) mice, was noted despite no significant differences in initial hepatic CD8(+) T cell IFN-gamma or TNF responses or in activation of intrahepatic cytotoxic lymphocytes cells capable of killing AdCMV-lacZ-infected fibroblast targets. In contrast, AdCMV-lacZ-infected hepatocyte targets were far more sensitive to killing by intrahepatic cytotoxic lymphocytes from B6.pfp(-/-) than from B6.gld mice, and residual levels of virus-specific killing of hepatocyte targets by FasL-defective B6.gld CTL were blocked by TNF inhibition. These results suggest that inherent resistance of hepatocytes to cytotoxicity mediated by perforin-dependent mechanisms leaves Fas/FasL-dependent, cell-mediated cytotoxicity as the major pathway for CTL-mediated killing of virally infected hepatocytes and accounts for the more prominent role of perforin-independent anti-viral mechanisms in immune responses in the liver.

Enhancement of MHC class I-stimulated alloresponses by TNF/TNF receptor (TNFR)1 interactions and of MHC class II-stimulated alloresponses by TNF/TNFR2 interactions.

In vivo TNF inhibition has been observed to ameliorate the disease process attributed to T cell-dependent immune responses such as those generated during graft-vs.-host disease. The present studies were designed to evaluate whether TNF/TNF receptor (TNFR)1 and TNF/TNFR2 interactions were involved in the generation of allospecific T cell responses. Splenic lymphocyte populations were obtained from TNFR1- or TNFR2-deficient B6 mice and from control B6 mice. These responder cells were cultured with irradiated MHC class II-disparate B6.C-H-2bm12 (bm12) or MHC class I-disparate B6.C-H-2bm1 (bm1) or irradiated syngeneic stimulator cells for 3 days before assay of [3H]thymidine incorporation. IL-2 levels of the mixed lymphocyte culture (MLC) supernatants were assessed by enzyme-linked immunosorbent assay. With MHC class II-disparate bm12 stimulator cells, a significant reduction in T cell proliferation was observed utilizing TNFR2-deficient CD4+ responder T cells, but not when using TNFR1 -deficient CD4+ responder T cells. A significant decrease in proliferation of TNFR1-deficient CD8+ responder cells, but not of TNFR2-deficient CD8 responder T cells was observed after stimulation with MHC class I-disparate bm1 stimulator cells. IL-2 levels were lower in MLC utilizing MHC class I stimulators and TNFR1-deficient responders or MHC class II stimulators and TNFR2-deficient responders. These results indicate that TNF/TNFR2 interactions promote MHC class II-stimulated alloresponses, while TNF/TNFR1 interactions promote MHC class I-stimulated alloresponses.

T-cell activation and differentiation are regulated by TNF during murine DBA/2-->B6D2F1 intestinal graft-versus-host disease.

Administration of a tumor necrosis factor (TNF) inhibitor-encoding adenoviral vector decreases the severity of colonic inflammation in a DBA/2-->B6D2F1 murine model of colonic graft-versus-host disease (GVHD). The present studies evaluated the effect of TNF blockade on the splenic and colonic T-cell responses. cDNA encoding an artificial fusion protein consisting of the extracellular domain of the human 55-kDa receptor for TNF fused to a mouse IgG heavy chain was subcloned into an E1a-deficient adenoviral vector. Following transfer of DBA/2 T cells and bone marrow cells into irradiated B6D2F1 mice, the mice then received either the control adenovirus or the TNF inhibitor-encoding adenovirus. Splenic and colonic lymphocytes were isolated, stained with anti-H-2b, anti-H-2d, anti-CD3, anti-CD4, anti-CD8, and anti-CD45RB antibodies, and analyzed by flow cytometry. Splenic and colonic lymphocyte cytokine profiles also were assessed. More colonic T cells of donor origin were isolated from the control adenovirus recipients than from recipients of the TNF inhibitor encoding adenovirus (P = .027). Fewer CD4+ and CD8+ T cells were observed in colon but not in the spleen in the TNF inhibitor recipients. Fewer CD45RBlow (memory) T cells were observed in the CD4+ colonic lymphocytes isolated from the TNF inhibitor recipients than from controls. Importantly, lower levels of interleukin-2(IL-2) and interferon-gamma (INF-gamma) but not of IL-4 were observed in the lamina propria lymphocyte RNA isolated from the TNF inhibitor recipients. Infiltration and expansion of donor T cells and T-cell activation in the colon appear to be regulated by TNF during murine DBA/2 --> B6D2F1 gut GVHD.

Diaphragmatic hernia seen as a late complication of laparoscopic cholecystectomy.

Laparoscopic surgery has emerged as the standard of care for the elective operative management of symptomatic gallbladder disease. The surgical literature is now beginning to accumulate sufficient case numbers that more clearly define the associated morbidity of this type of surgery. This article reports an instance of iatrogenic injury to the right muscular hemidiaphragm and subsequent hernia after laparoscopic cholecystectomy.

Investigation of the role of postnatal testosterone in the expression of sex differences in behavior in infant rhesus macaques (Macaca mulatta).

In several primate species, males have been shown to exhibit a surge in circulating testosterone during the early postnatal period. This surge has been postulated to play a role in the development of sex differences in behavior. In this study, the role of postnatal testosterone in infant behavioral development was investigated in socially living rhesus macaques. Seven male infants were treated with a GnRH agonist, avorelin, from the first week of life onwards. Ten female infants were exposed to testosterone by implantation of capsules containing testosterone. The behavioral development of these and control infants was recorded from birth to 6 months of age. The sexually dimorphic patterns of play and mounting were not affected by manipulation of postnatal testosterone in either male or female infants. Similarly, most mother-infant interactions were not affected by the hormonal manipulation of infants. Mothers of testosterone-treated females were found to take more responsibility for moving into and out of arm's reach of their infants than mothers of some other groups of infants; however, this measure did not normally differ between mothers of male and female infants. Manipulation of the postnatal testosterone surge does significantly affect penile growth and development, but does not affect the expression of infant sex differences in behavior nor greatly affect the development of the mother-infant relationship in rhesus macaques.

Tumor necrosis factor inhibitor ameliorates murine intestinal graft-versus-host disease.

BACKGROUND & AIMS: Transfer of T helper cells from DBA/2 mice to irradiated allogeneic B6D2F1 mice leads to development of colonic graft-versus-host disease with pathological features of inflammatory bowel disease. To examine the role of tumor necrosis factor (TNF) in graft-versus-host disease enteropathy, an adenoviral vector encoding a TNF inhibitor protein was administered. METHODS: Irradiated B6D2F1 mice were infused with DBA/2 bone marrow and spleen cells. Mice then received either a control beta-galactosidase-encoding adenovirus or an adenovirus encoding a TNF inhibitor, composed of the extracellular domain of the human 55-kilodalton TNF receptor linked to the murine immunoglobulin G1 heavy chain. Mucosal permeability to sucralose and colonic histology were assessed 14 and 25 days after transplantation. RESULTS: Less diarrhea was observed in DBA/2 --> B6D2F1 mice expressing the TNF inhibitor, and colonic sections from these mice had significantly less inflammation and epithelial cell abnormalities. In TNF inhibitor recipients, mucosal permeability to sucralose was similar to that in nonirradiated control mice and significantly less than in recipients of the control adenovirus. CONCLUSIONS: TNF inhibition decreases the severity of enteropathy in the DBA/2 --> B6D2F1 murine model of colonic graft-versus-host disease.

Lymphoid hyperplasia, CD45RBhigh to CD45RBlow T-cell imbalance, and suppression of Type I diabetes mellitus result from TNF blockade in NOD-->NOD-scid adoptive T cell transfer.

Sustained antibody-mediated inhibition of tumor necrosis factor (TNF) activity offers protection against Type I (insulin-dependent) diabetes mellitus in non-obese diabetic (NOD) mice. The mechanism of this effect, however, has remained obscure: TNFalpha might be required for the development of specific immune responses to islet antigens or it could directly participate in destruction of beta cells. In this study, autoimmune destruction of beta cells was initiated in NOD-severe combined immunodeficient (scid) mice by transfer of NOD splenic T-cells to induce diabetes. The blockade of TNFalpha activity was achieved during a narrow window of time after transfer. Transient inhibition of TNFalpha greatly reduced the number of islet lymphocytes and the incidence of diabetes in recipients of prediabetic NOD spleen cells. Protection extended beyond the interval of effective TNF blockade. Furthermore, the protective effect was only observed if cells were obtained from 6-week-old donors. The suppression of autoimmunity was reversible in the context of adoptive transfer as indicated by the transfer of splenocytes from the primary recipient to a second NOD-scid host led to a diabetic outcome. The blockade of TNFalpha was accompanied by a considerable increase in spleen size and doubling of the total splenocyte count, suggesting that TNFalpha might normally eliminate a transplanted T-cell subset within the recipients. Further analysis showed an increase in the absolute count of CD4 + T cells and pronounced distortion of the CD45RBhigh to CD45RBlow ratio, with a relative augmentation in the CD45RBlow count in the spleen. TNFalpha appears to regulate the number and subtype distribution of a transplanted T cell population.

Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels.

Short-term regulation of insulin gene transcription by glucose.

Whereas short-term regulation of insulin biosynthesis at the level of translation is well accepted, glucose-dependent transcriptional control is still believed to be a long-term effect occurring after more than 2 hr of glucose stimulation. Because pancreatic beta cells are exposed to elevated glucose levels for minutes rather than hours after food uptake, we hypothesized the existence of a short-term transcriptional control. By studying the dynamics of newly synthesized (prepro)insulin RNA and by employing on-line monitoring of gene expression in single, insulin-producing cells, we were able to provide convincing evidence that insulin gene transcription indeed is affected by glucose within minutes. Exposure of insulinoma cells and isolated pancreatic islets to elevated glucose for only 15 min resulted in a 2- to 5-fold elevation in (prepro)insulin mRNA levels within 60-90 min. Similarly, insulin promoter-driven green fluorescent protein expression in single insulin-producing cells was significantly enhanced after transient glucose stimulation. Thus, short-term signaling, such as that involved in insulin secretion, also may regulate insulin gene transcription.

Adenovirus-mediated blockade of lymphotoxin-beta inhibits the induction of contact sensitivity in mice.

Lymphotoxin-beta is a newly recognized member of the tumor necrosis factor ligand family. Recent studies have suggested a role for this cytokine in delayed-type hypersensitivity responses. To determine whether lymphotoxin-beta contributes to the development of contact sensitivity, we utilized an inhibitor protein that can effectively block binding of lymphotoxin-beta to its receptor. An adenoviral vector was created that encodes for a lymphotoxin-beta inhibitor protein consisting of the extracellular domain of the lymphotoxin-beta receptor fused to IgG heavy chain. Intravenous injection of the recombinant virus into BALB/c mice yielded plasma levels of inhibitor protein > 500 micrograms that persisted for 1 week. Mice treated in this manner were compared with control animals injected with adenovirus encoding beta-galactosidase, with respect to their ability to mount contact sensitivity responses to epicutaneously applied dinitro-fluorobenzene. Mice transduced with the lymphotoxin-beta inhibitor prior to the induction of contact sensitivity showed significantly suppressed ear swelling responses. By contrast, mice treated with the lymphotoxin-beta inhibitor prior to the elicitation of contact sensitivity showed no change in ear swelling responses in comparison to controls. These findings indicate that lymphotoxin-beta plays an important role in the afferent phase of the contact sensitivity response.

Adenoviral vectors given intravenously to immunocompromised mice yield stable transduction of the colonic epithelium.

BACKGROUND & AIMS: Adenoviral vectors have been used for gene transfer in the liver but not for gene transfer in intestinal tissue. The aim of this study was to show that in selectively immunocompromised mice injected intravenously with a recombinant adenovirus, higher levels of a reporter gene are expressed in the colon than in the liver. METHODS: Adenovirus encoding beta-galactosidase was injected intravenously in lethally irradiated B6D2F1 mice that had received syngeneic B6D2F1 bone marrow and spleen cell transplants, in athymic mice, in mice treated with 2-chlorodeoxyadenosine, or in normal mice. Enzymatic assays and polymerase chain reaction analysis were performed on colonic tissue obtained months after transduction. Colonic tissues were also stained for beta-galactosidase. RESULTS: Intravenous adenoviral administration yielded long-term expression of a foreign gene in liver and colonic epithelium in transiently immunocompromised recipients. Histological analysis suggested that stem cell transfection and integration of the foreign gene may have occurred insofar as crypts and colonic epithelial cells in immunocompromised animals stained positive for beta-galactosidase months after virus administration. In polymerase chain reaction analysis, the transverse and distal colon of syngeneic bone marrow transplant recipients showed long-term retention of beta-galactosidase gene. CONCLUSIONS: Long-term transduction of colonic epithelial cells is observed after administration of adenoviral vectors by an intravenous route in selectively immunocompromised mice.

Effects of altering testosterone in early infancy on social behaviour in captive yearling rhesus monkeys.

Thirteen male and twenty female rhesus monkeys (Macaca mulatta), aged 9-12 months, living as members of long term captive social groups, were observed in order to quantify sex differences in a variety of behaviour patterns. Six males had been treated with a GnRH agonist (Meterelin: M) during their first six postnatal months, in order to block the surge of testosterone which occurs at this time. Ten females had been treated with testosterone (T) during their first six months, in order to mimic the postnatal T surge seen in males. The remaining 7 males and 10 females acted as control subjects. Marked sex differences were measured in frequencies of play and socio-sexual behaviour in these juvenile monkeys. However, neither M nor T treatments produced any significant changes in frequencies of these behaviour patterns. Although M-treated males showed a tendency (p < 0.1) to groom others for longer periods than control males, and control females tended to spend more time alone than T-treated females, we were unable to measure any significant (p < 0.05) effects of either M- or T-treatment upon affiliative behaviour in juvenile rhesus monkeys. We conclude, therefore, that the postnatal T surge in male rhesus monkeys does not affect development of sexually dimorphic and associated patterns of behaviour. Presumably, organisational effects of T upon these behaviour patterns must be completed before birth in this species.

Inositol lipid-mediated signalling in response to endothelin and ATP in the mammalian testis.

The testis is a complex organ in which local control is achieved by signalling between its constituent cells. Herein we describe the responses of cultured rat testicular cells and a mouse Sertoli cell-line to stimulation by endothelin and ATP, and elsewhere we have shown that rat peritubular myoid cells possess phosphoinositidase C-coupled V1a-vasopressin receptors identical to those of liver (Howl, J. et al, 1995, Endocrinology 136: 2206-2213). 1. Peritubular myoid cells from pre-pubertal rats responded through ETA receptors with PtdIns(4,5)P2 hydrolysis [EC50 for endothelin-1 (ET-1) approximately 0.4 nM], elevation of intracellular [Ca2+], and tyrosine phosphorylation of a variety of cellular proteins. They also showed enhanced adenylate cyclase activity, with an EC50 for ET-1 of approximately 3 nM, also through ETA receptors. Pharmacological elevation of [cAMP] did not immediately change the ET-1-stimulated formation of inositol phosphates, but attenuated the response after several hours. 2. Pre-pubertal rat Sertoli cells showed no detectable responses to ET-1, but responded to FSH with elevated [cAMP] and to ATP with PtdIns(4,5)P2 hydrolysis. PtdIns(4,5)P2 hydrolysis was equally responsive to ATP and UTP, and so appears to be activated by P2U-purinergic receptors. This response was enhanced by protein kinase C inhibition and attenuated by PKC activation. 3. Despite its lack of effect on rat Sertoli cells in primary culture, ET-1 provoked PtdIns(4,5)P2 hydrolysis in the TM4 murine Sertoli cell line (EC50 approximately 0.6 nM), and this response was negatively regulated by protein kinase C activation. 5. No receptor-stimulated activation of phosphoinositase C was detected in 'germ cell' populations, but the non-specific G protein activator A1F4-provoked inositol phosphate accumulation in these cells, so demonstrating their potential to respond through yet to be identified G protein-coupled receptors with phosphoinositidase C activation. 6. Immunoblotting studies showed the presence in rat testis of phosphoinositidase C-beta 1 and the alpha-subunits(s) of the G-protein(s) Gq and/or G11. These studies show that testicular myoid and Sertoli cells use at least three G protein-coupled receptors (V1a-vasopressins, ETA-endothelin and P2U-purinergic) to signal through phosphoinositidase C activation, that ET-1 can activate multiple signalling pathways in myoid cells, and that the ET-1-stimulated phosphoinositidase C responses of myoid and Sertoli cells have different regulatory characteristics.

CD27-CD27 ligand/CD70 interactions enhance alloantigen-induced proliferation and cytolytic activity in CD8+ T lymphocytes.

To study the role of CD27-CD27 ligand (CD27L)/CD70 interactions in the generation of murine allospecific T cell responses, SF9 cells or cell membranes expressing recombinant human CD70 were added to in vitro MLC containing C57BL/6 (H-2b) responder cells and class I and II MHC disparate H-2b/d stimulator cells. Alloantigen-specific CTL generation, CD8+ T cell proliferation, and levels of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase activity were enhanced in the presence of human CD27L/CD70 expressed on SF9 cell membranes. Enhancement of CD8+ T cell responses occurred in the absence of any discernible effects on CD4+ T cell proliferation or IL-2 responses. Additional studies demonstrated that CD27L/CD70-expressing membranes enhanced proliferative responses to class I MHC differences but had no effect on proliferative responses to class II MHC disparities. Enhancement of allospecific CTL generation was also observed when CD27L/CD70-expressing membranes were added only during the last 24 to 48 h of 5-day MLC. Thus, the present studies suggest that CD27-CD27L/CD70 interactions can selectively enhance differentiation of Ag-specific CD8+ T cell effector mechanisms under conditions in which Th cell responses are not altered.

The use of methylphenidate for cognitive decline associated with HIV disease.

OBJECTIVE: Complaints of cognitive changes are often expressed by patients at all stages of HIV infection. Such changes include decreased memory and attention span, diminished concentration, apathy, and "slowing." Methylphenidate (MPD) has been used in several clinical studies in men with late-stage HIV disease in an attempt to ameliorate these difficulties. The objectives of this review article are to review salient psychopharmacological characteristics of MPD and to describe the research and clinical literature supporting the use of MPD in patients at all stages of HIV infection. METHODS: Seven studies, case reports, or abstracts from International Conferences on AIDS were available in the English literature through August, 1993, directly addressing the use of MPD in patients with HIV disease. Twenty-nine papers were reviewed for pharmacokinetic data, eighteen for safety and side effects issues, and seventeen for relevant contributions from the neuropsychological testing literature. RESULTS: Studies in clinical settings have used doses ranges from 10-90 mg. per day in two or three divided doses with reportedly good results in improving both affective and cognitive symptoms associated with HIV disease. Side effects have been relatively mild and patient satisfaction with treatment has been high. However, no studies have been conducted in early stage HIV disease, where a significant minority of patients have similar complaints in the absence of clinically apparent immunosuppression. Likewise, placebo-controlled, dose-finding studies in AIDS patients are entirely lacking, and no studies in women with HIV disease and cognitive changes have been published. CONCLUSIONS: In spite of these important research short-comings, clinical experience with MPD treatment of cognitive changes in men with HIV/AIDS is consistent with the notion that this medication holds significant promise to improve the quality of life for persons living with HIV/AIDS. Controlled studies to test this hypothesis are warranted.

Divergent effects of epidermal growth factor and calcipotriol on human rectal cell proliferation.

Vitamin D may protect against colorectal cancer by reducing cell proliferation and inducing differentiation. By contrast, epidermal growth factor (EGF) stimulates cell proliferation and may encourage gastrointestinal mucosal healing. This study investigated the effect of a synthetic vitamin D analogue, calcipotriol, and EGF on human rectal epithelial cell proliferation in patients with familial adenomatous polyposis (FAP). In addition, a new technique to measure the cell cycle time is described. Sigmoidoscopic biopsy specimens were obtained from 14 patients with FAP. Tissue was established in organ culture, with or without the addition of EGF (n = 8), or calcipotriol (n = 6). Proliferation was determined using (a) metaphase arrest to measure the crypt cell production rate, (b) native mitotic index, and (c) the growth fraction using PC10 antibody. EGF receptor expression was shown using a polyclonal antibody AP12E. Calcipotriol reduced crypt cell production rate by 52% from mean (SEM) 5.29 (1.18) to 2.56 (0.80) cells/crypt/hour (p < 0.01) and EGF increased crypt cell production rate by 102% from 3.62 (0.59) to 7.33 (0.90) cells/crypt/hour (p < 0.05), and this tissue expressed the EGF receptor. The growth fraction was 48.40 (4.0)%, and the native mitotic index 1.08 (0.14)%. The cell cycle time was estimated as 94.5 hours and the time for mitosis as one hour. Thus, calcipotriol and EGF have divergent effects on human rectal mucosal proliferation.

Characterisation of a novel Ca2+ pump inhibitor (bis-phenol) and its effects on intracellular Ca2+ mobilization.

Bis-phenol, a phenolic antioxidant, is an inhibitor of sarcoplasmic reticulum (SR), endoplasmic reticulum (ER) and plasma membrane Ca2+ ATPases. The concentration of bis-phenol giving half-maximal inhibition of the SR Ca(2+)-ATPase is 2 microM. On binding to the SR Ca(2+)-ATPase it shifts the E2 to E1 transition towards the E2 state and slows the transition between E2 to E1. Bis-phenol completely inhibits Ca(2+)-dependent ATP hydrolysis and Ca2+ uptake by rat cerebellar microsomes at a concentration of 30 microM. The plasma membrane Ca(2+)-ATPase is also completely inhibited at similar concentrations, however, the Na+/K(+)-ATPase is only marginally affected. Other inhibitors of the ER Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibit Ca2+ uptake by approximately 75%. Bis-phenol therefore inhibits all types of ER Ca(2+)-ATPases present in cerebellum. This inhibitor is also able to mobilize Ca2+ from intracellular Ca2+ stores, including those sensitive to InsP3, in intact HL-60 cells.

Inositol 1,4,5-trisphosphate-, GTP-, arachidonic acid- and thapsigargin-mediated intracellular calcium movement in PANC-1 microsomes.

Inositol 1,4,5-trisphosphate (IP3)-, GTP-, arachidonic acid- and thapsigargin-mediated Ca2+ release from endoplasmic reticulum (ER)-enriched microsomes was studied in a PANC-1 cell line. IP3 maximally caused an approximately 20% release of actively accumulated Ca2+. This effect was completely blocked by heparin. In the presence of 3% polyethylene glycol (PEG), GTP maximally discharged about 60% of Ca2+ from the microsomes. This effect involved a GTP hydrolytic process, not the IP3-activated Ca2+ channel. Arachidonic acid maximally released approximately 80% of Ca2+ from PANC-1 microsomes. Metabolites of arachidonic acid did not appear to be involved in arachidonic acid-mediated Ca2+ release. However, other fatty acids also induced similar releasing effects suggesting that arachidonic acid-induced Ca2+ release appeared to be non-specific. Thapsigargin was shown to inhibit Ca2+ accumulation into and induce Ca2+ release from PANC-1 microsomes. The thapsigargin-releasable Ca2+ pool included the IP3- or arachidonic acid-sensitive pool. Studies on liposomes suggested that both arachidonic acid and thapsigargin did not exert either a Ca2+ ionophore-like or a membrane detergent-like effect. The present results have provided evidence for the existence of multiple non-mitochondrial Ca2+ pools in PANC-1 cells. These Ca2+ pools could be released by various Ca2+ mediators via different mechanisms.