Targeted delivery of cytotoxic proteins to prostate cancer via conjugation to small molecule urea-based PSMA inhibitors.

Prostate cancer cells are characterized by a remarkably low proliferative rate and the production of high levels of prostate-specific proteases. Protein-based toxins are attractive candidates for prostate cancer therapy because they kill cells via proliferation-independent mechanisms. However, the non-specific cytotoxicity of these potent cytotoxins must be redirected to avoid toxicity to normal tissues. Prostate-Specific Membrane Antigen (PSMA) is membrane-bound carboxypeptidase that is highly expressed by prostate cancer cells. Potent dipeptide PSMA inhibitors have been developed that can selectively deliver and concentrate imaging agents within prostate cancer cells based on continuous PSMA internalization and endosomal cycling. On this basis, we conjugated a PSMA inhibitor to the apoptosis-inducing human protease Granzyme B and the potent Pseudomonas exotoxin protein toxin fragment, PE35. We assessed selective PSMA binding and entrance into tumor cell to induce cell death. We demonstrated these agents selectively bound to PSMA and became internalized. PSMA-targeted PE35 toxin was selectively toxic to PSMA producing cells in vitro. Intratumoral and intravenous administration of this toxin produced marked tumor killing of PSMA-producing xenografts with minimal host toxicity. These studies demonstrate that urea-based PSMA inhibitors represent a simpler, less expensive alternative to antibodies as a means to deliver cytotoxic proteins to prostate cancer cells.

The severity and associated comorbidities of retinopathy of prematurity among micro-premature infants with birth weights less than 750 grams.

OBJECTIVES: To evaluate the characteristics and comorbidities associated with ROP in micro-premature infants and their results. METHODS: This is a retrospective chart review involving multiple intensive care units in Central Texas from 2011 to 2016. Infants were included if birth weight (BW) was≤750 g with confirmed ROP by the International Classification of Retinopathy of Prematurity (ICROP). Neonates were examined and treated with laser ablation or intravitreal ranibizumab (IVR) with subsequent laser treatment, guided by fluorescein angiography, if met treatment criteria defined as type 1 ROP by the Early Treatment of Retinopathy of Prematurity standards. Time to regression was defined clinically. Results were analyzed using chi-squared test. RESULTS: 100 neonates were included in the study. Mean BW was 599 grams and mean gestational age was 24.2 weeks. Forty neonates were classified as type 1 ROP and therefore required intervention; of them 21 received laser alone and 19 required IVR with subsequent laser. Only 2 patients received more than one IVR injection. None of the patients progressed to stage 4 or 5 ROP. CONCLUSIONS: Despite such low birth weights, none of these neonates progressed to stage 4 or 5 ROP likely because of prompt examination and treatment with laser or with IVR and subsequent laser. IVR might serve as a bridge to laser in type 1 ROP allowing some retinal vessel development prior to definitive laser treatment.

Human tissue legislation: listening to the professionals.

The controversies in Bristol, Alder Hey and elsewhere in the UK surrounding the removal and retention of human tissue and organs have led to extensive law reform in all three UK legal systems. This paper reports a short study of the reactions of a range of health professionals to these changes. Three main areas of ethical concern were noted: the balancing of individual rights and social benefit; the efficacy of the new procedures for consent; and the helpfulness for professional practice of the new legislation and regulation. Recognition of these concerns may help in forging a new partnership between professionals and patients and their families.

Toxicity of the antimicrobial compound triclosan and formation of the metabolite methyl-triclosan in estuarine systems.

Triclosan, a commonly used antimicrobial compound, has been measured in aquatic systems worldwide. This study exposed marine species to triclosan to examine effects primarily on survival and to investigate the formation of the degradation product, methyl-triclosan, in the estuarine environment. Acute toxicity was assessed using the bacterium Vibrio fischeri, the phytoplankton species Dunaliella tertiolecta, and three life stages of the grass shrimp Palaemonetes pugio. P. pugio larvae were more sensitive to triclosan than adult shrimp or embryos. Acute aqueous toxicity values (96 h LC50) were 305 microg/L for adult shrimp, 154 microg/L for larvae, and 651 microg/L for embryos. The presence of sediment decreased triclosan toxicity in adult shrimp (24 h LC50s were 620 microg/L with sediment, and 482 microg/L without sediment). The bacterium was more sensitive to triclosan than the grass shrimp, with a 15 min aqueous IC50 value of 53 microg/L and a 15 min spiked sediment IC50 value of 616 microg/kg. The phytoplankton species was the most sensitive species tested, with a 96 h EC50 value of 3.55 microg/L. Adult grass shrimp were found to accumulate methyl-triclosan after a 14-day exposure to 100 microg/L triclosan, indicating formation of this metabolite in a seawater environment and its potential to bioaccumulate in higher organisms. Triclosan was detected in limited surface water sampling of Charleston Harbor, SC at a maximum concentration of 0.001 microg/L, substantially lower than the determined toxicity values. These findings suggest triclosan poses low acute toxicity risk to estuarine organisms; however, the potential for chronic, sublethal, and metabolite effects should be investigated.

Clustering of urokinase receptors (uPAR; CD87) induces proinflammatory signaling in human polymorphonuclear neutrophils.

Leukocytes use urokinase receptors (uPAR; CD87) in adhesion, migration, and proteolysis of matrix proteins. Typically, uPAR clusters at cell-substratum interfaces, at focal adhesions, and at the leading edges of migrating cells. This study was undertaken to determine whether uPAR clustering mediates activation signaling in human polymorphonuclear neutrophils. Cells were labeled with fluo-3/AM to quantitate intracellular Ca2+ ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by Ab cross-linking. Aggregating uPAR induced a highly reproducible increase in [Ca2+]i (baseline to peak) of 295 +/- 37 nM (p = 0.0002). Acutely treating cells with high m.w. urokinase (HMW-uPA; 4000 IU/ml) produced a response of similar magnitude but far shorter duration. Selectively aggregating uPA-occupied uPAR produced smaller increases in [Ca2+]i, but saturating uPAR with HMW-uPA increased the response to approximate that of uPAR cross-linking. Cross-linking uPAR induced rapid and significant increases in membrane expression of CD11b and increased degranulation (release of beta-glucuronidase and lactoferrin) to a significantly greater degree than cross-linking control Abs. The magnitude of degranulation correlated closely with the difference between baseline and peak [Ca2+]i, but was not dependent on the state of uPA occupancy. By contrast, selectively cross-linking uPA-occupied uPAR was capable of directly inducing superoxide release as well as enhancing FMLP-stimulated superoxide release. These results could not be duplicated by preferentially cross-linking unoccupied uPAR. We conclude that uPAR aggregation initiates activation signaling in polymorphonuclear neutrophils through at least two distinct uPA-dependent and uPA-independent pathways, increasing their proinflammatory potency (degranulation and oxidant release) and altering expression of CD11b/CD18 to favor a firmly adherent phenotype.

Transcatheter arterial embolization in blunt hepatic trauma.

Patients with blunt hepatic injury can safely be managed nonoperatively if they show hemodynamic stability. Transcatheter arterial embolization (TAE) is a useful adjunct in the treatment of patients who show evidence of continued hemorrhage or who have pooling of contrast material on computed tomography (CT). In these patients, TAE may reduce transfusion requirements and allow healing of the injury without operation. Complications are uncommon and are usually managed nonoperatively.

Pharmacists' readiness to assess consumers' over-the-counter product selections.

OBJECTIVE: To measure and explain pharmacists' readiness to adopt a proposed new standard for assessing consumers' over-the-counter product selections. DESIGN: Cross-sectional, descriptive study based on the theoretical underpinnings of the Transtheoretical Model of Change. A questionnaire was used for data collection. Sections included: (1) pharmacists' readiness to engage in a proposed standard of practice; (2) the positive and negative aspects of doing so; (3) pharmacists' agreement with current legislative status of pharmacist-only products; and (4) demographics. SETTING: Community pharmacy. PARTICIPANTS: Community pharmacists in one Canadian province. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Pharmacists' readiness to adopt a proposed new standard of care. RESULTS: Response rate was 70.6%. Most respondents were staff pharmacists in independent pharmacies. The majority were not ready to adopt the proposed new behavior--57.6% were in the precontemplation stage. Age, sex, year of graduation, workload, and size of town/city had no significant effect on readiness for change. As expected, precontemplators showed less overall support for keeping pharmacist-only agents behind the counter. Positive and negative beliefs about the proposed standard varied across stages. CONCLUSION: Any initiatives to increase pharmacist involvement in assessing consumer nonprescription product selections must acknowledge that pharmacists differ in their degree of readiness for change.

Urokinase receptor (CD87) aggregation triggers phosphoinositide hydrolysis and intracellular calcium mobilization in mononuclear phagocytes.

Leukocytes utilize urokinase receptors (uPAR; CD87) in adhesion, migration, and matrix proteolysis. uPAR aggregate at cell-substratum interfaces and at leading edges of migrating cells, so this study was undertaken to determine whether uPAR aggregation is capable of initiating activation signaling. Monocyte-like U937 cells were labeled with fluo-3-acetoxymethyl ester to quantitate intracellular Ca2+ concentrations ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by mAb cross-linking. uPAR aggregation induced highly reproducible increases in [Ca2+]i of 103.0 +/- 10.9 nM (p < 0.0001) and >3-fold increases in cellular d-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Similar increases in [Ca2+]i were also elicited by uPAR aggregation in human monocytes, but cross-linking a control IgG2a had no effect on [Ca2+]i. Selectively cross-linking uPA-occupied uPAR with an anti-uPA mAb produced smaller increases in [Ca2+]i, but fully saturating uPAR with exogenous uPA enhanced the [Ca2+]i response to equal the effect of aggregating uPAR directly. Increased [Ca2+]i was inhibited by thapsigargin, herbimycin A, and U73122, but only partially reduced by low extracellular [Ca2+], indicating that uPAR aggregation increases [Ca2+]i by activating phospholipase C through a tyrosine kinase-dependent mechanism, generating Ins(1,4,5)P3 and releasing Ca2+ from Ins(1,4, 5)P3-sensitive intracellular stores. Cross-linking the beta2 integrin CR3 could not duplicate the effect of uPAR cross-linking, and uPAR-triggered Ca2+ mobilization was not blocked by anti-CR3 mAbs. These results indicate that uPAR aggregation initiates phosphoinositide hydrolysis by mechanisms that are not strictly dependent on associated uPA or CR3.

Pressures and rewards of working in community mental health teams.

Community mental health teams (CMHTs) are widely regarded as the mainstay of community services, bringing health and social services professionals together to target people with severe mental health problems. Heather Harper and Edana Minghella report the findings of a recent survey looking at the pressures and rewards of working in these community-based, multi-disciplinary teams.

Suppression of vestibular nystagmus by forced convergence in normal human subjects.

BACKGROUND: It has been suggested that the principal mechanism of nystagmus suppression in the nystagmus blockage syndrome is either adduction of the eye or convergence. We examined this issue using the nystagmus of the vestibulo-ocular reflex (VOR) as a model. METHODS: A motorized, computer-controlled rotary chair was used to produce VOR in darkness, using either sinusoidal or velocity step stimulation. Left eye position was monitored and horizontal slow-phase eye velocity was calculated. Subjects were cued to converge or perform other gaze tasks. RESULTS: Convergence suppressed nystagmus. With sinusoidal stimulation, nystagmus was nearly extinguished in extreme lateroversion, probably due to mechanical tethering of the eye. However, VOR gain suppression of 47% during convergence was observed even when the monitored eye was close to primary position. With velocity step stimulation, nystagmus was nearly extinguished at moderate angles of adduction. CONCLUSIONS: Convergence is sufficient to suppress nystagmus, without vision and without regard to whether the eye is adducted.

The role of small intestinal antigen-presenting cells in the induction of T-cell reactivity to soluble protein antigens: association between aberrant presentation in the lamina propria and oral tolerance.

The oral administration of soluble protein antigen results in profound immunological tolerance. However, the tissue location and function of antigen-presenting cells (APC) that stimulate this response remain unclear. We have hypothesized that the properties of cells presenting antigen to naive T cells within the gut are involved, and therefore gut APC should stimulate T-cell responses with different characteristics to those induced by other APC. To test this, we studied in vitro primary T-cell responses following presentation of soluble protein antigen by cells from the Peyer's patches (PPC) and lamina propria (LPC) of the murine small intestine and the spleen (SPLC). Each APC population stimulated antigen-specific proliferative responses with similar anamnestic characteristics; however, analysis of the cytokines produced revealed marked differences. Whereas SPLC stimulated the balanced production of T-helper type 1 (Th1) and Th2 cytokines, PPC induced a profile consistent with the provision of T-cell help for IgA production. Interestingly, presentation of antigen by LPC stimulated high levels of interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) in the absence of other cytokines [interleukin-2 (IL-2), IL-4, IL-5]. Evidence from analysis of cell activation and division within the cultures suggested that this profile may result from the preferential activation of CD8+ T cells by LPC; however, the lack of conventional CD4+ T-cell cytokines indicated a defect in the normal function of these cells. Adoptive transfer of antigen-pulsed LPC to syngeneic animals abrogated the induction of delayed-type hypersensitivity (DTH) responsiveness, which followed a subsequent conventional antigen challenge further suggesting a role for lamina propria APC in tolerance induction.

Botulinum A exotoxin for glabellar folds: a double-blind, placebo-controlled study with an electromyographic injection technique.

BACKGROUND: Botulinum A exotoxin (BTX) has been used successfully to treat a variety of hyperkinetic movement disorders. BTX is also capable of reducing hyperkinetic facial lines including prominent glabellar frown lines. OBJECTIVE: The purposes of this study were to (1) confirm the efficacy of BTX in a double-blind, placebo-controlled investigation; (2) evaluate the use of an electromyogram attached to the injection needle to confirm intramuscular corrugator placement of the BTX; and (3) determine the optimum direction injection technique. METHODS: Length and depth of glabellar frown lines were measured before treatment and 4 and 12 weeks after injection of 10 units of BTX or saline solution. RESULTS: Patients treated with BTX had a highly significant reduction in depth and length of glabellar frown lines compared with control subjects. CONCLUSION: BTX appears to be effective and safe for reduction of glabellar frown lines.

The human amnion is a site of MHC class Ib expression: evidence for the expression of HLA-E and HLA-G.

The expression of HLA class I Ag by term human amnion epithelial cells was investigated. In immunostaining and FACS analysis, mAb to monomorphic class I Ag reacted extensively with amnion cells, whereas polymorphic mAb reactivity was more limited and variable. Further studies were conducted on amnion cell preparations containing negligible contaminants. Northern analysis with use of locus-specific probes demonstrated that amnion expresses two class Ib genes, HLA-E and HLA-G. Radio-immunoprecipitation with use of monomorphic mAb identified two fully glycosylated cell surface class I H chains of 44 and 41 kDa; polymorphic mAbs failed to immunoprecipitate the 41-kDa product, although 44-kDa products, typical of class Ia Ag, were identified in some preparations. Class I H chains were isolated from amnion by affinity chromatography. Microsequencing revealed that the first nine residues of the N-terminus of the 41-kDa product aligned perfectly only with HLA-E. Overall, amnion at term appears to express class Ib Ag with limited class Ia Ag. HLA-G is therefore expressed in two extrafetal epithelia: amnion and trophoblast. Identification of the class Ib protein HLA-E in amnion epithelium may have implications for preterm labor that can be associated with infection of the placental membranes.

Therapist contributions to psychotherapeutic assimilation: an alternative to the drug metaphor.

A psychotherapist's verbal interventions may be understood as promoting a client's eventual improvement by facilitating developmental change processes within the client. This approach is an alternative to the traditional search for statistical links between aggregates of therapist interventions and global outcome measures. Our approach employs models of clients' assimilation of problematic experiences within problem domains and therapists' implementation of theoretically specified aims. In an empirical illustration, one client's change within a particular problem domain and its links with therapist interventions were assessed qualitatively across the course of brief psychodynamic-interpersonal treatment.

Resolving a challenge to the therapeutic relationship: a single-case study.

The selection of a case of demonstrated change using a quantitative criterion preceded an exploration of possible change mechanisms in therapy. Between-session variations in the therapeutic relationship revealed by a quantitative measure prompted an intensive qualitative investigation of in-session events. Within these events, the study focused on challenges to the therapeutic relationship and their subsequent resolution. Understanding the resolution of such challenges to the relationship was guided by a rational model derived from the treatment rationale. A tentative account of the process of change is offered, and case replications are required to confirm and extend the findings.