In vivo and in vitro properties of STX2484: a novel non-steroidal anti-cancer compound active in taxane-resistant breast cancer.

BACKGROUND: STX2484 is a novel non-steroidal compound with potent anti-proliferative activity. These studies aimed to identify STX2484's mechanism of action, in vivo efficacy and activity in taxane-resistant breast cancer models. METHODS: Effects of STX2484 and paclitaxel on proliferation, cell cycle and apoptosis were assessed in vitro in drug-resistant (MCF-7(DOX)) and non-resistant cells (MCF-7(WT)). STX2484 efficacy in ßIII tubulin overexpression in MCF-7 cells was also determined. Anti-angiogenic activity was quantified in vitro by a co-culture model and in vivo using a Matrigel plug assay. An MDA-MB-231 xenograft model was used to determine STX2484 efficacy in vivo. RESULTS: STX2484 is a tubulin disruptor, which induces p53 expression, Bcl2 phosphorylation, caspase-3 cleavage, cell cycle arrest and apoptosis. In addition, STX2484 is a potent anti-angiogenic agent in vitro and in vivo. In breast cancer xenografts, STX2484 (20 mg kg(-1) p.o.) suppressed tumour growth by 84% after 35 days of daily dosing, with limited toxicity. In contrast to paclitaxel, STX2484 efficacy was unchanged in two clinically relevant drug-resistant models. CONCLUSIONS: STX2484 is an orally bioavailable microtubule-disrupting agent with in vivo anti-angiogenic activity and excellent in vivo efficacy with no apparent toxicity. Crucially, STX2484 has superior efficacy to paclitaxel in models of clinical drug resistance.

New structural insights provide a different angle on steroid sulfatase action.

A central part of human sulfation pathways is the spatially and temporally controlled desulfation of biologically highly potent steroid hormones. The responsible enzyme - steroid sulfatase (STS) - is highly expressed in placenta and peripheral tissues, such as fat, colon, and the brain. The shape of this enzyme and its mechanism are probably unique in biochemistry. STS was believed to be a transmembrane protein, spanning the Golgi double-membrane by stem region formed by two extended internal alpha-helices. New crystallographic data however challenge this view. STS now is portraited as a trimeric membrane-associated complex. We discuss the impact of these results on STS function and sulfation pathways in general and we hypothesis that this new STS structural understanding suggests product inhibition to be a regulator of STS enzymatic activity.

BCRP expression does not result in resistance to STX140 in vivo, despite the increased expression of BCRP in A2780 cells in vitro after long-term STX140 exposure.

The anti-proliferative and anti-angiogenic properties of the endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2), are enhanced in a series of sulphamoylated derivatives of 2-MeOE2. To investigate possible mechanisms of resistance to these compounds, a cell line, A2780.140, eightfold less sensitive to the 3,17-O,O-bis-sulphamoylated derivative, STX140, was derived from the A2780 ovarian cancer cell line by dose escalation. Other cell lines tested did not develop STX140 resistance. RT-PCR and immunoblot analysis demonstrated that breast cancer resistance protein (BCRP) expression is dramatically increased in A2780.140 cells. The cells are cross-resistant to the most structurally similar bis-sulphamates, and to BCRP substrates, mitoxantrone and doxorubicin; but they remain sensitive to taxol, an MDR1 substrate, and to all other sulphamates tested. Sensitivity can be restored using a BCRP inhibitor, and this pattern of resistance is also seen in a BCRP-expressing MCF-7-derived cell line, MCF-7.MR. In mice bearing wild-type (wt) and BCRP-expressing tumours on either flank, both STX140 and mitoxantrone inhibited the growth of the MCF-7wt xenografts, but only STX140 inhibited growth of the MCF-7.MR tumours. In conclusion, STX140, a promising orally bioavailable anti-cancer agent in pre-clinical development, is highly efficacious in BCRP-expressing xenografts. This is despite an increase in BCRP expression in A2780 cells in vitro after chronic dosing with STX140.

2-Methoxyoestradiol-3,17-O,O-bis-sulphamate and 2-deoxy-D-glucose in combination: a potential treatment for breast and prostate cancer.

Drug combination therapy is a key strategy to improve treatment efficacy and survival of cancer patients. In this study the effects of combining 2-methoxyoestradiol-3,17-O,O-bis-sulphamate (STX140), a microtubule disruptor, with 2-deoxy-D-glucose (2DG) were assessed in MCF-7 (breast) and LNCaP (prostate) xenograft models in vivo. In mice bearing MCF-7 xenografts, daily p.o. administration of STX140 (5 mg kg(-1)) resulted in a 46% (P<0.05) reduction of tumour volume. However, the combination of STX140 (5 mg kg(-1) p.o.) and 2DG (2 g kg(-1) i.p.) reduced tumour volume by 76% (P<0.001). 2-Methoxyoestradiol-3,17-O,O-bis-sulphamate also reduced tumour vessel density. 2-Deoxy-D-glucose alone had no significant effect on tumour volume or vessel density. A similar benefit of the combination treatment was observed in the LNCaP prostate xenograft model. In vitro the degree of inhibition of cell proliferation by STX140 was unaffected by oxygen concentrations. In contrast, the inhibition of proliferation by 2DG was enhanced under hypoxia by 20 and 25% in MCF-7 and LNCaP cells, respectively. The combination of STX140 and 2DG in LNCaP cells under normoxia or hypoxia inhibited proliferation to a greater extent than either compound alone. These results suggest that the antiangiogenic and microtubule disruption activities of STX140 may make tumours more susceptible to inhibition of glycolysis by 2DG. This is the first study to show the benefit of combining a microtubule disruptor with 2DG in the two most common solid tumours.

The in vivo properties of STX243: a potent angiogenesis inhibitor in breast cancer.

The steroidal-based drug 2-ethyloestradiol-3,17-O,O-bis-sulphamate (STX243) has been developed as a potent antiangiogenic and antitumour compound. The objective of this study was to ascertain whether STX243 is more active in vivo than the clinically relevant drug 2-methoxyoestradiol (2-MeOE2) and the structurally similar compound 2-MeOE2-3,17-O,O-bis-sulphamate (STX140). The tumour growth inhibition efficacy, antiangiogenic potential and pharmacokinetics of STX243 were examined using four in vivo models. Both STX243 and STX140 were capable of retarding the growth of MDA-MB-231 xenograft tumours (72 and 63%, respectively), whereas no inhibition was observed for animals treated with 2-MeOE2. Further tumour inhibition studies showed that STX243 was also active against MCF-7 paclitaxel-resistant tumours. Using a Matrigel plug-based model, in vivo angiogenesis was restricted with STX243 and STX140 (50 and 72%, respectively, using a 10 mg kg(-1) oral dose), thereby showing the antiangiogenic activity of both compounds. The pharmacokinetics of STX243 were examined at two different doses using adult female rats. The compound was orally bioavailable (31% after a single 10 mg kg(-1) dose) and resistant to metabolism. These results show that STX243 is a potent in vivo drug and could be clinically effective at treating a number of oncological conditions.

Steroid metabolism in breast cancer.

The endocrine system and its steroids have long been thought to be instrumental in the etiology of breast cancer. A large proportion of cancerous breast tissues have been shown to express estrogen (ER), androgen (AR) and progesterone (PR) receptors. It is through these receptors that steroid hormones can exert their mitogenic effects. The local biosynthesis of estrogens is believed to play an integral part in the development of hormone-dependent breast cancer and recent studies on the use of inhibitors to block this steroid production has yielded an improvement of prognosis in breast cancer patients. Consequently, the understanding of the enzymes involved in the synthesis and metabolism of estrogens in breast cancer is paramount to treating this malignancy. This review examines the biological and clinical relevance of three key endocrine enzymes: steroid sulfatase (STS), aromatase (Arom), and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type-1. The importance of the over expression and increased activity of these enzymes in breast tissue and on breast cancer is discussed. Importantly, the intratumoral biosynthesis of estrogens is examined in detail. The effects of new inhibitors of these enzymes on the growth of hormone-dependent breast cancer will also be investigated. First and second generation STS inhibitors and third generation aromatase inhibitors are showing significant promise, whereas inhibitors for 17beta-HSD type-1 are still at an early stage. However, such endocrine therapy that is currently being explored has shown promising results for patients with hormone-dependent breast cancer.

NNT is a key regulator of adrenal redox homeostasis and steroidogenesis in male mice.

Nicotinamide nucleotide transhydrogenase, NNT, is a ubiquitous protein of the inner mitochondrial membrane with a key role in mitochondrial redox balance. NNT produces high concentrations of NADPH for detoxification of reactive oxygen species by glutathione and thioredoxin pathways. In humans, NNT dysfunction leads to an adrenal-specific disorder, glucocorticoid deficiency. Certain substrains of C57BL/6 mice contain a spontaneously occurring inactivating Nnt mutation and display glucocorticoid deficiency along with glucose intolerance and reduced insulin secretion. To understand the underlying mechanism(s) behind the glucocorticoid deficiency, we performed comprehensive RNA-seq on adrenals from wild-type (C57BL/6N), mutant (C57BL/6J) and BAC transgenic mice overexpressing Nnt (C57BL/6JBAC). The following results were obtained. Our data suggest that Nnt deletion (or overexpression) reduces adrenal steroidogenic output by decreasing the expression of crucial, mitochondrial antioxidant (Prdx3 and Txnrd2) and steroidogenic (Cyp11a1) enzymes. Pathway analysis also revealed upregulation of heat shock protein machinery and haemoglobins possibly in response to the oxidative stress initiated by NNT ablation. In conclusion, using transcriptomic profiling in adrenals from three mouse models, we showed that disturbances in adrenal redox homeostasis are mediated not only by under expression of NNT but also by its overexpression. Further, we demonstrated that both under expression or overexpression of NNT reduced corticosterone output implying a central role for it in the control of steroidogenesis. This is likely due to a reduction in the expression of a key steroidogenic enzyme, Cyp11a1, which mirrored the reduction in corticosterone output.

Localization of the binding regions of a murine monoclonal anti-factor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues threonine351-serine365 of the factor VIII heavy chain.

We have localized the binding region of a previously described monoclonal anti-factor VIII (FVIII) inhibitory antibody (C5) to amino acid residues Thr351-Ser365 of the thrombin-generated 54-kD fragment of the heavy chain of FVIII. Synthetic FVIII peptides were examined for the ability to competitively inhibit the binding of C5 to FVIII in an ELISA system. The synthetic FVIII peptide Thr351-Ser365 blocked C5 binding to FVIII in a dose-dependent manner in this system. Two other synthetic FVIII peptides, Asn340-Glu354 and Glu342-Asp356, which partially overlapped Thr351-Ser365, also blocked C5 binding to FVIII. Blocking of C5 binding with these peptides, however, required much greater concentrations (greater than 100 times stronger) than that required for Thr351-Ser365. The Thr351-Ser365 peptide also neutralized the FVIII inhibitory activity of C5 in plasma. A human FVIII inhibitor (anti-FVIII heavy chain alloantibody) was also partially neutralized by Thr351-Ser365. Thr351-Ser365 lies between a thrombin cleavage site (Arg372) and an activated protein C cleavage site (Arg336) and may be at or near a region of functional importance in the expression of FVIII procoagulant activity.

Intracellular trafficking of factor VIII to von Willebrand factor storage granules.

In plasma, von Willebrand factor (vWf) associates with Factor VIII (FVIII); however, the site at which these proteins first interact has not been defined. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) causes a rapid, concomitant elevation in plasma levels of both vWf and FVIII, suggesting the existence of a DDAVP-releasable storage pool for both proteins. To determine whether vWf and FVIII can associate intracellularly and colocalize to storage vesicles, we transfected AtT-20 cells with vWf and FVIII expression plasmids. FVIII alone was not detectable within storage granules; however, transfection of vWf cDNA into the same cell caused FVIII to alter its intracellular trafficking and to undergo granular storage, colocalizing to the vWf-containing granules. In contrast, colocalization of FVIII was not observed when these cells were transfected with plasmids encoding defective FVIII-binding vWf mutants. Transfection of bovine endothelial cells with FVIII further demonstrated vesicular storage of FVIII with vWf in Weibel-Palade bodies. Since gene therapy of hemophilia A may ultimately target endothelium or hematopoietic stem cells, the interaction between vWf and FVIII within a secretory cell is important. Thus, vWf can alter the intracellular trafficking of FVIII from a constitutive to a regulated secretory pathway, thereby producing an intracellular storage pool of both proteins.

Factor VIII structure and function.

The relatively recent ability to obtain highly purified factor VIII (FVIII) preparations from plasma products, the cloning of the FVIII gene, and the expression of recombinant FVIII have provided the basis for significant advancements in the understanding of the structure-function relationships of FVIII. Evaluation of the molecular structure of FVIII has revealed the presence of domains of significant internal amino acid sequence homology as well as homology with similar structural domains of factor V. Specific proteolytic cleavage sites have been identified in the molecule and the use of site directed mutagenesis has identified those proteolytic cleavage sites required for the activation of FVIII. Deletion and substitution variants of FVIII as well as the precise epitope mapping of FVIII antibodies which inhibit the procoagulant function of the protein or its binding to von Willebrand factor have provided insight into the identification of regions of FVIII which are required for normal function.

A perspective on the use of FVIII concentrates and cryoprecipitate prophylactically in surgery or therapeutically in severe bleeds in patients with von Willebrand disease unresponsive to DDAVP: results of an international survey. On behalf of the Subcommittee on von Willebrand Factor of the Scientific and Standardization Committee of the ISTH.

An international registry was established by the Subcommittee on von Willebrand Factor of the SSC/ISTH on the treatment of patients with types of von Willebrand disease (vWD) unresponsive to DDAVP infusion. Data was collected on 76 surgical events in 64 patients from 19 treatment centers. Thirty-three non-mucosal, 12 mucosal, 10 orthopedic and 21 dental procedures were reported. In the 76 surgical events, 14 cases prophylactically received cryoprecipitate while 62 received factor VIII (FVIII) concentrate. Surgical hemostasis was reported as satisfactory, good, or excellent in 75 of the 76 cases. Post-infusion bleeding times were measured in only three of 14 surgical events treated with cryoprecipitate. All three cases had a reduction but not correction of the bleeding time. The post-infusion bleeding time was measured in 27 of 62 cases in which FVIII concentrates were used. The bleeding time time was normalized in 15, reduced but not normalized in eight, and not changed from baseline in four. Data was also collected from 16 treatment centers on 50 serious bleeding events in 35 patients. These included 19 gastrointestinal, 15 other mucosal, four central nervous system, seven orthopedic, and five other mucosal, four central nervous system, seven orthopedic, and five other bleeds. Eleven cases received cryoprecipitate and 39 received FVIII concentrate as primary therapy. The efficacy of treatment was considered good or excellent in 49 of 50 cases. Post-infusion bleeding times were measured in only 15 of the 50 reported bleeding events. The post-infusion bleeding time was normalized in six, decreased but not normalized in eight, and not changed from baseline in one. In this retrospective survey, FVIII concentrates were subjectively as efficacious as cryoprecipitate in both the surgical setting and for the treatment of severe bleeds in patients with types of vWd unresponsive to DDAVP. Since the bleeding time was monitored during therapy in only a minority of these cases, a definitive relationship between the efficacy of therapy and normalization or reduction of the bleeding time in these two clinical settings cannot be established from this study. A prospective study on the use of FVIII and/or vWf concentrates in these clinical settings is necessary to address this issue.

The reproductive health of women with von Willebrand Disease unresponsive to DDAVP: results of an international survey. On behalf of the Subcommittee on von Willebrand Factor of the Scientific and Standardization Committee of the ISTH.

An international registry was established on the reproductive health of women with types of von Willebrand disease (vWd) unresponsive to DDAVP. Data was collected on 44 women from 16 treatment centers in nine countries. Severe menorrhagia requiring blood product therapy occurred at least once in 80% of the women for whom data was reported. Most of the reported episodes occurred prior to the diagnosis of vWd and/or the use of oral contraceptive (OC) therapy. OC therapy was clinically effective in the treatment of chronic menorrhagia in 22 of 25 (88%) women treated. Two of the women, however, were unable to tolerate chronic OC use and a third became refractory to treatment. Hysterectomy was performed in 10 of the 44 women (23%). The reported indication for six of the procedures was menorrhagia. Seventy percent were performed at two treatment centers, suggesting different thresholds for the performance of the procedure. There were 69 pregnancies reported in 31 of the 44 women. Fifteen of the pregnancies resulted in spontaneous abortions. The incidence of miscarriage was 22% and appeared clustered, with 10 of 15 of the miscarriages occurring in just four of the women.

The polymerase chain reaction with sequence specific primers for the detection of the factor V mutation associated with activated protein C resistance.

The prevalence of the Factor V (FV) mutation associated with activated protein C resistance (FV Leiden) and its significance as a genetic risk factor for venous thrombosis have necessitated the development of a simple, rapid, and accurate assay for its detection. The polymerase chain reaction with sequence specific primers (PCR-SSP) provides a powerful technique for the discrimination of alleles resulting from single base substitutions. PCR amplification was performed using a sense primer complementary to both FV alleles coupled with either of two antisense allele specific primers, one complementary to the normal FV allele and one complementary to the FV Leiden allele. PCR conditions were developed that favored amplification only in the case of perfect complementation between template DNA and allele specific primer. The FV genotype was assigned based on whether or not each allele specific primer set produced an amplified product. Assignment of genotypes correlated 100% with those determined by the method of PCR amplification followed by Mn1I digestion. PCR-SSP allows the rapid and accurate identification of carriers of the Factor V Leiden mutation by a simple PCR reaction without the need for the usual post-amplification specificity step.

A synthetic factor VIII peptide of eight amino acid residues (1677-1684) contains the binding region of an anti-factor VIII antibody which inhibits the binding of factor VIII to von Willebrand factor.

The monoclonal anti-factor VIII (FVIII) antibody C4 has previously been reported to inhibit the binding of purified FVIII to immobilized von Willebrand factor (vWF). The binding area of C4 was identified to be within fifteen amino acid residues (1670-1684) based on the ability of a synthetic FVIII peptide consisting of amino acid residues 1670-1684 to completely inhibit the binding of C4 to FVIII. We now report the further localization of the binding region of C4 to within eight amino acid residues (1677-1684) of FVIII light chain. Nine new overlapping FVIII peptides were synthesized based on the amino acid sequence of the acidic region of FVIII light chain and tested, along with seven previously tested peptides, for the ability to inhibit C4 binding to FVIII in an ELISA assay. Three synthetic FVIII peptides 1670-1684, 1675-1690, and 1677-1684 demonstrated dose dependent inhibition of C4 binding to FVIII. The three reactive peptides contain residues 1677-1684 in common. Since C4 can completely inhibit the binding of FVIII to vWF, this report further localizes an eight amino acid residue region of FVIII which may be important in the mediation of vWF binding.

Fine mapping of monoclonal antibody epitopes on human von Willebrand factor using a recombinant peptide library.

A recombinant human von Willebrand factor (vWF) cDNA fragment library was constructed in lambda gt11 for the localization of anti-vWF monoclonal antibody epitopes. Twelve of 21 monoclonal antibodies screened identified epitopes expressed in lambda gt11 as beta-galactosidase fusion proteins. By sequence analysis, these antigenic determinants were localized to segments ranging from 17 to 105 amino acids in length. Four epitopes apparently shared by more than one antibody were identified, suggesting the presence of immuno-dominant epitopes within vWF. Monoclonal antibody C3, which blocks factor VIII (FVIII) binding to vWF, bound to the same epitope previously identified by a second monoclonal antibody which also blocks this function, suggesting that this region may be at or near the vWF/FVIII binding domain. Three antibodies recognize the same region within the vWF A2 repeat. Mutations near this region appear to be responsible for Type IIA von Willebrand's disease. The co-localization of these antibodies suggests that this domain might be exposed on the surface of vWF, consistent with its apparent increased sensitivity to plasma proteases.

Phenotypic correction of activated protein C resistance following orthotopic liver transplantation.

A 43-year-old white female underwent orthotopic liver transplantation in 1992 for cirrhosis related to primary sclerosing cholangitis. Pre-transplantation protein S (PS) studies revealed a discrepancy between PS activity and free PS antigen consistent with type II PS deficiency. Since the presence of activated protein C (APC) resistance has been reported to interfere with PS activity assays resulting in an apparent type II PS deficiency, we retrospectively tested a pre-transplantation frozen plasma sample for APC resistance. The sample was found to have an abnormal APC resistance ratio (APCR-R) of 1.71. Follow up testing 2 1/2 years post-transplantation revealed correction of the APC resistance phenotype (normalization of the APCR-R to 2.79). Analysis of DNA extracted from lymphocytes revealed the patient to be heterozygous for the FV mutation associated with APC resistance (FV Leiden). Hereditary APC resistance was confirmed by family studies which revealed the presence of APC resistance and heterozygous FV Leiden in her son. Although the patient's post-transplantation plasma FV is normal, her platelet FV remains heterozygous for FV Leiden. To what extent, if any, platelet FV Leiden in the absence of plasma FV Leiden may contribute to a predisposition to thrombosis is unknown.

A murine monoclonal anti-factor VIII inhibitory antibody and two human factor VIII inhibitors bind to different areas within a twenty amino acid segment of the acidic region of factor VIII heavy chain.

The factor VIII (FVIII) binding regions of the monoclonal anti-FVIII inhibitory antibody C5 and a human FVIII inhibitor antibody have previously been reported to be contained within amino acid residues 351-365 of FVIII. Localization of the binding regions of these two antibodies was based on their reactivity with four synthetic FVIII peptides. Nineteen synthetic FVIII peptides spanning the entire acidic region of the FVIII heavy chain have now been evaluated for the ability to inhibit the binding of C5 to FVIII in an ELISA assay. The smallest peptide tested that inhibited C5 binding to FVIII consisted of residues 351-361. Those peptides that were able to inhibit C5 binding in the ELISA assay were also able to neutralize the FVIII inhibitory activity of C5 in plasma. The FVIII inhibitory activity of two human FVIII inhibitor antibodies was also partially neutralized by peptides from this region. Evaluation of the pattern of peptides reactive with the three antibodies indicates that the binding regions of these antibodies are in very close proximity to each other, but are not identical. Their respective binding regions are contained within residues 351-361 (C5), 354-362 (inhibitor 1), and 342-354 (inhibitor 2). These results suggest that this 20 amino acid segment of the acidic region of the heavy chain of FVIII may be functionally important in the expression of FVIII procoagulant activity.

Crisis resource management among strangers: principles of organizing a multidisciplinary group for crisis resource management.

Patient safety depends on the skills, vigilance, and judgment of trained individuals working as members of a clinical team that includes anesthesiologists, surgeons, nurses, and technicians. Now, as never before, safe outcome depends both on better knowledge and better management. This requires organization of caregivers, who may be strangers from diverse disciplines, into teams. One can drill an individual to work safely alone. One can rehearse a series of scenarios with small groups (who regularly work together) to improve performance. But what does one do with an unrehearsed group, called together in an emergency from several different disciplines, usually including Anesthesia. These people may not know each other, their roles, their special skills, and may even be hazy about each other's goals. Rapid organization of such an ad hoc team becomes a critical priority where patient safety is at stake. The way by which such an ad hoc team from several disciplines can rapidly be helped to function effectively together is by teaching all the "strangers" the principles of Crisis Resource Management. These principles are not as well-presented in a written text or lecture format, as one cannot introduce the sense of urgency that emotionally charges and changes the impact. We believe the best teacher is experience gained in a realistic simulated environment using a model driven, full human simulator. This simulated environment is safe for both patient and trainee.

Low dose epidural lidocaine/sufentanil is effective for outpatient lithotripsy.

Lumbar epidural analgesia was administered to 60 ASA class 1 & 2 patients with 3 ml test dose of 1.5% lidocaine and bolus of 20 ml of 0.5% lidocaine containing 0.5 microgram/kg sufentanil. Bilateral decreased lumbar cold perception was accepted as evidence of analgesia despite persisting pinprick sensation in thoracic dermatomes. Oxygen saturation (SpO2), respiratory rate, cardiovascular parameters and leg muscle strength were monitored throughout and until 1 hour afterwards. Midazolam provided light sedation and atropine bradycardia control. Verbal communication was maintained. ESWL could start within 6-10 minutes of bolus, with analgesia adequate in 86% of patients, the rest being "rescued" with 5-10 ml 0.5% lidocaine or analgesic doses (20-30 mg IV) of ketamine. Leg weakness developed in 14%, with 1 patient fully paralyzed. All resolved within 1 hour. Topical urethral analgesia was used in males where cystoscopy preceded ESWL. Phenylephrine was required once for nild systolic hypotension, otherwise blood pressures were stable. Two of 4 patients experiencing pruritus needed naloxone relief. Itching appeared in skin recovering from sensory block while visceral analgesia persists. Excessive respiratory depression was not seen.

School-based clinics: overcoming the obstacles.

Obstacles to the establishment of school-based clinics are investigated to determine possible strategies for the future. The current social environment necessitates a health care model that provides increased access and improved outcomes for children in particular. The advanced registered nurse practitioner is an integral part of the school-based clinic. Although opposition to school-based clinics has been well organized in the past, proponents are learning new ways to deal with this challenge.