A pilot evaluation of the expanded prostate cancer index composite for clinical practice (EPIC-CP) tool in Ontario.

PURPOSE: To introduce the EPIC-CP symptom screening tool in routine ambulatory cancer care, and to evaluate its acceptability and perceived usefulness from the perspective of patients and clinicians. METHODS: Eligible prostate cancer patients from four cancer centres were recruited (November 2014-June 2015) from radiation or surgical oncology clinics. A physician and/or health care professional reviewed the EPIC-CP results as part of the clinical encounter. Patient experience with the tool was evaluated using a nine-item Patient Exit Survey (PES). Clinician experience was evaluated through semi-structured qualitative interviews. Patient and clinician results were compared to identify common themes. RESULTS: A total of 333 patients were enrolled, of whom, 287 completed the PES. Most patients had one clinical encounter, although the number of EPIC-CP assessments ranged from 1 to 11 per patient, for a total of 937 EPIC-CP questionnaires completed. Item completion rates were high (91-100%), with items addressing sexual health among the lowest (91-92%). On the PES, most patients (70%) agreed with the item: "Completing this questionnaire helped me tell the clinicians about how I have been feeling". Thematic analysis from clinician interviews revealed that the EPIC-CP captures essential prostate-specific effects that facilitated person-centred communication and customization of interventions. Targeted clinical education and patient resources were seen as necessary for uptake. CONCLUSIONS: EPIC-CP was generally endorsed by clinicians and patients. The implementation of a disease-specific measure in place of a generic symptom screening tool has the potential to improve the quality of the clinical encounter and provide outcome measures for further health services research. Provincial implementation of this tool as a standard of care is recommended.

A mixed-method evaluation of nurse-led community-based supportive cancer care.

GOALS OF WORK: The study purpose was to evaluate a nurse-led supportive care clinical case management program in the community using multi-methods to delineate care processes prior to outcome evaluation. MATERIALS AND METHODS: Multiple data sources including program service records, chart reviews and interviews with nurses and key interdisciplinary informants were used to identify population served (coverage and reach), processes of care (implementation), and providers' perceptions of the effectiveness of the nurse-led program (reaction). MAIN RESULTS: The program provided care to over 700 cancer patients in a 1-year period. Nurse-led support interventions were focused on direct care inclusive of teaching/coaching for symptom management, counseling and support, and mobilization of services through system navigation based on an initial comprehensive assessment of supportive care needs. CONCLUSIONS: Nurse-led models of supportive care have the potential to reduce unmet supportive care needs, improve continuity of care, and overall health-related quality of life that should be tested in future trials.

Identification of enzymes homologous to isocitrate dehydrogenase that are involved in coenzyme B and leucine biosynthesis in methanoarchaea.

Two putative Methanococcus jannaschii isocitrate dehydrogenase genes, MJ1596 and MJ0720, were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze the NAD- and NADP-dependent oxidative decarboxylation of DL-threo-3-isopropylmalic acid, threo-isocitrate, erythro-isocitrate, and homologs of threo-isocitrate. Neither enzyme was found to use any of the isomers of isocitrate as a substrate. The protein product of the MJ1596 gene, designated AksF, catalyzed the NAD-dependent decarboxylation of intermediates in the biosynthesis of 7-mercaptoheptanoic acid, a moiety of methanoarchaeal coenzyme B (7-mercaptoheptanylthreonine phosphate). These intermediates included (-)-threo-isohomocitrate [(-)-threo-1-hydroxy-1,2, 4-butanetricarboxylic acid], (-)-threo-iso(homo)(2)citrate [(-)-threo-1-hydroxy-1,2,5-pentanetricarboxylic acid], and (-)-threo-iso(homo)(3)citrate [(-)-threo-1-hydroxy-1,2, 6-hexanetricarboxylic acid]. The protein product of MJ0720 was found to be alpha-isopropylmalate dehydrogenase (LeuB) and was found to catalyze the NAD-dependent decarboxylation of one isomer of DL-threo-isopropylmalate to 2-ketoisocaproate; thus, it is involved in the biosynthesis of leucine. The AksF enzyme proved to be thermostable, losing only 10% of its enzymatic activity after heating at 100 degrees C for 10 min, whereas the LeuB enzyme lost 50% of its enzymatic activity after heating at 80 degrees C for 10 min.

(R)-citramalate synthase in methanogenic archaea.

The Methanococcus jannaschii gene MJ1392 was cloned, and its protein product was hyperexpressed in Escherichia coli. The resulting protein was purified and shown to catalyze the condensation of pyruvate and acetyl coenzyme A, with the formation of (R)-citramalate. Thus, this gene (cimA) encodes an (R)-citramalate synthase (CimA). This is the first identification of this enzyme, which is likely involved in the biosynthesis of isoleucine.

Alpha-keto acid chain elongation reactions involved in the biosynthesis of coenzyme B (7-mercaptoheptanoyl threonine phosphate) in methanogenic Archaea.

The biochemistry of the 13 steps involved in the conversion of alpha-ketoglutarate and acetylCoA to alpha-ketosuberate, a precursor to the coenzymes coenzyme B (7-mercapto heptanoylthreonine phosphate) and biotin, has been established in Methanosarcina thermophila. These series of reactions begin with the condensation of alpha-ketoglutarate and acetylCoA to form trans-homoaconitate. The trans-homoaconitate is then hydrated and dehydrated to cis-homoaconitate with (S)-homocitrate serving as an intermediate. Rehydration of the cis-homoaconitate produces (-)-threo-isohomocitrate [(2R,3S)-1-hydroxy-1,2, 4-butanetricarboxylic acid], which undergoes a NADP+-dependent oxidative decarboxylation to produce alpha-ketoadipate. The resulting alpha-ketoadipate then undergoes two consecutive sets of alpha-ketoacid chain elongation reactions to produce alpha-ketosuberate. In each of these sets of reactions, it has been shown that the homologues of cis-homoaconitate, homocitrate, and (-)-threo-isohomocitrate serve as intermediates. The protein product of the Methanococcus jannaschii MJ0503 gene aksA (AksA) was found to catalyze the condensation of alpha-ketoglutarate and acetylCoA to form trans-homoaconitate. This gene product also catalyzed the condensation of alpha-ketoadipate or alpha-ketopimelate with acetylCoA to form, respectively, the (R)-homocitrate homologues of (R)-2-hydroxy-1,2,5-pentanetricarboxylic acid and (R)-2-hydroxy-1,2, 6-hexanetricarboxylic acid. The alpha-ketosuberate resulting from this series of reactions then undergoes a nonoxidative decarboxylation to form 7-oxoheptanoic acid, a precursor to coenzyme B, and an oxidative decarboxylation to form pimelate, the precursor to biotin. Of the 13 intermediates in this pathway, eight have not previously been reported as occurring in biological systems.

D-erythro-neopterin biosynthesis in the methanogenic archaea Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH.

The steps in the biosynthetic transformation of GTP to 7,8-dihydro-D-erythro-neopterin (H2neopterin), the precursor to the modified folates found in the methanogenic archaea, has been elucidated for the first time in two members of the domain Archaea. In Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH, it has been demonstrated that H2neopterin 2':3'-cyclic phosphate is an intermediate in this conversion. In addition, the formation of the pterin ring of the H2neopterin 2':3'-cyclic phosphate is catalyzed not by a single enzyme, as is known to occur with GTP cyclohydrolase I in the Eucarya and Bacteria, but rather by two or more enzymes. A 2,4,5-triamino-4(3H)-pyrimidinone-containing molecule, most likely 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, has been identified as an intermediate in the formation of the H2neopterin 2':3'-cyclic phosphate. Synthetic H2neopterin 2':3'-cyclic phosphate was found to be readily hydrolyzed by cell extracts of M. thermophila via the H2neopterin 3'-phosphate to H2neopterin, a known precursor to the pterin portion of methanopterin.

Decreased frequency of functional natural interferon-producing cells in peripheral blood of patients with the acquired immune deficiency syndrome.

Deficient in vitro production of interferon-alpha (IFN-alpha) in response to herpes simplex virus (HSV) occurs in patients infected with the human immunodeficiency virus (HIV), with the most deficient responses associated with opportunistic infections (OI). The peripheral blood mononuclear cells (PBMC) which produce IFN-alpha in response to HSV are light density, HLA-DR+ cells lacking any unique surface markers and have been termed "natural interferon-producing cells" (NIPC). In this study, IFN-alpha responses were measured and the ELISpot assay was utilized to determine the frequency of NIPC in response to HSV. As expected, HIV-infected patients had depressed IFN-alpha production. In the ELISpot assay, healthy controls had a mean frequency of 1:703 NIPC among PBMC; each NIPC made approximately 2 international units (IU) of IFN-alpha. HIV-infected patients on average had fourfold less NIPC than controls and produced 1 IU IFN-alpha/NIPC; the plaque size for patient samples was often smaller than that for controls. NIPC frequency and IFN-alpha production were lowest in patients with a history of OI. In conclusion, deficient IFN-alpha production by AIDS patients results from reductions in both the frequency and the activity of NIPC, probably reflecting a gradual turning off of IFN-alpha production.

Peripheral blood of AIDS patients contains cells capable of providing accessory function for the natural killer cell-mediated, lysis of herpes simplex virus-infected targets despite low interferon-alpha production.

We have previously demonstrated that in vitro production of interferon-alpha (IFN-alpha) in response to herpes simplex virus (HSV) by peripheral blood mononuclear cells PBMCs from patients infected with the human immunodeficiency virus (HIV-1) decreases dramatically with disease progression, with extremely low levels of IFN-alpha preceding and predictive of opportunistic infections. Natural killer (NK) lysis, however, was found to decay later in disease and often was within normal limits even when IFN-alpha production was severely compromised. The NK lysis of HSV-infected fibroblasts (HSV-FS) is dependent on an HLA-DR+ accessory cell (AC) population that shares the phenotype of the predominant IFN-alpha-producing cell (IPC) population. To determine whether there is a correlation between AC activity and IFN-alpha production in these patients, we tested the ability of PBMCs from AIDS patients to provide AC help to NK cells from heterologous donors. While NK cells were highly sensitive to gamma irradiation, AC activity was relatively radioresistant. Therefore, NK cells from healthy donors were depleted of HLA-DR+ ACs and added to irradiated PBMCs from either healthy or AIDS donors to test for the function of ACs in the irradiated populations. Irradiated cells from AIDS patients were found to provide normal AC activity despite decreased IFN-alpha production in the majority of the patients. We failed to observe NK augmenting activity in supernatants of irradiated PBMCs from IFN-deficient patients that had been stimulated with HSV-FS.(ABSTRACT TRUNCATED AT 250 WORDS)

Immediate-early gene expression is sufficient for induction of natural killer cell-mediated lysis of herpes simplex virus type 1-infected fibroblasts.

Herpes simplex virus type 1 (HSV-1)-infected human fibroblast (HSV-FS) targets are susceptible to lysis by natural killer (NK) cells, whereas uninfected FS are resistant to lysis. Studies were undertaken to determine the mechanism of this preferential susceptibility. HSV-FS were not intrinsically less stable than FS, as determined by a 51Cr release assay under hypotonic shock in the presence of rat granule cytolysin and by sensitivity to anti-human leukocyte antigen class I antibody plus complement. Single-cell assays in agarose demonstrated that although similar numbers of large granular lymphocytes bound to the HSV-FS and FS targets, the conjugates with HSV-FS were lysed at a much higher frequency than those with FS. These results suggested that both targets are bound by the NK cells but only the HSV-FS were able to trigger lysis. The requirement for active virus expression was demonstrated by failure of emetine-treated HSV-FS targets or targets infected with UV-inactivated HSV to be lysed by NK effectors. To evaluate the role of viral glycoproteins in conferring susceptibility to lysis, Fab were prepared from HSV-1-seropositive sera; these Fab were unable to block lysis of the HSV-FS. Furthermore, incubation in phosphonoacetic acid failed to reduce NK(HSV-FS) activity despite sharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissive temperature were lysed as well as or better than targets infected with wild-type virus, indicating that HSV immediate-early gene product expression is sufficient for conferring susceptibility to lysis. We conclude that expression of nonstructural viral proteins or virally induced cellular gene products early in the course of infection rather than structural glycoproteins is required for NK lysis of HSV-FS targets.

Natural killer-mediated lysis of some but not all HSV-1- or VSV-infected targets requires the participation of HLA-DR-positive accessory cells.

Natural killer (NK)-mediated lysis of herpes simplex virus type 1-infected fibroblasts (HSV-FS) has been previously shown to require the co-operation of CD16-positive NK cells and an HLA-DR-positive accessory cell population. In contrast, lysis of K562 tumour cells requires the presence of only the Leu-11-positive cells. In the current study, targets of different morphologies, both virally infected and non-infected, were tested in an attempt to dissect out which target characteristics determine the need for accessory cell participation for NK-mediated lysis. Effector populations were obtained through antibody plus complement (C) depletions of subpopulations of human peripheral blood mononuclear cells using anti-HLA-DR+C (accessory cell depleted) or anti-CD16+C (NK depleted). The subpopulations were tested both alone and mixed together for their ability to mediate target lysis. Although NK-mediated lysis of most HSV-infected targets required the presence of HLA-DR-positive accessory cells, there was one set of exceptions. Lysis of the non-adherent Epstein-Barr virus (EBV)-transformed lymphoblastoid lines HSV-Raji, HSV-ARH and HSV-CCRF demonstrated only partial accessory cell dependence. All infected adherent cell lines were accessory cell dependent. In contrast, none of the adherent or non-adherent non-infected targets tested required the presence of DR-positive accessory cells for killing. Therefore, the presence of virus was an indicator of accessory cell dependence for NK-mediated kill except in the cases where HSV-infected EBV-transformed targets were used. Assay times of 4 hr versus 14 hr were conducted to determine if the kinetics of kill of various targets correlated with the requirement for accessory cells. A substantial percentage of the total lysis seen at 14 hr occurred within 4 hr for accessory cell independent lysis of the non-infected targets. In contrast, accessory cell-dependent kill of infected targets usually required longer incubation time for substantial lysis to occur, and correlated with interferon (IFN) production. NK-mediated lysis of vesicular stomatitis virus-infected fibroblasts required the presence of both the CD16- and HLA-DR-positive subpopulations, extending the role of DR-positive cells in NK-mediated killing beyond herpes virally infected targets.

CTL: virus control cells first and cytolytic cells second? DNA fragmentation, apoptosis and the prelytic halt hypothesis.

It is usually presumed that cytotoxic T lymphocytes (CTL) stop viral replication by lysing infected cells before a full virus yield has been assembled. Unlike complement-mediated lysis, however, CTL induce apoptosis, including fragmentation of target cell DNA. Why should CTL do this? Here, Eric Martz and Donna Howell suggest that since the major function of CTL appears to be control of viruses, CTL may be able to halt viral replication without inducing rapid lysis. It may be more useful to think of CTL as virus control cells rather than as cytolytic cells.

Low calcium concentrations support killing by some but not all cytolytic T lymphocytes, and reveal inhibition of a postconjugation step by calcium antagonists.

Previous findings support the prediction that drugs which antagonize the action of calcium should inhibit cytolytic T lymphocyte (CTL)-mediated killing without inhibiting the formation of Ag-specific CTL-target cell conjugates. This would contrast with other CTL-inhibiting drugs, nearly all of which inhibit conjugate formation. Testing this prediction, we found that two calcium channel blockers (verapamil and ruthenium red) inhibit killing only when the extracellular calcium concentration is low (100 microM), and, as predicted, do not inhibit conjugate formation. Surprisingly, the esterase inhibitor N alpha-p-tosyl-L-lysine choloromethylketone also inhibited killing without inhibiting conjugate formation. Unexpectedly, we found that the amount of calcium required by CTL varies by four-fold or more. CTL produced in vivo, or by a single Ag stimulation cycle in vitro, require more than 130 microM calcium for optimal killing, whereas 30 microM suffices for CTL primed in vivo plus boosted in vitro. The rate of admission of calcium into the cytoplasm by physiologic channels did not appear to be the limiting factor for the former type of CTL. Recent findings indicate that allospecific CTL produced in vivo may lack cytoplasmic granules, and may kill by an unidentified mechanism distinct from the exocytosis of granules prominent in CTL lines or clones maintained in vitro. The differences in calcium requirements reported here may reflect differences in mechanisms of killing.

Nuclear disintegration induced by cytotoxic T lymphocytes. Evidence against damage to the nuclear envelope of the target cell.

CTL and NK cells induce nuclear disintegration in their target cells. This phenomenon, which is seen as extensive fragmentation and solubilization of target cell DNA, is not seen with most other means of inducing cytolysis, including antibody- and complement-mediated cytolysis. We have previously shown that the degree of DNA solubilization is dependent upon the nature of the target cell. We here investigate the possibility that CTL induce, in all targets, damage to the nuclear envelope, which in turn leads to nuclear disintegration in only some of them. We reasoned that damage to the nuclear envelope would render nuclear DNA more accessible to exogenous DNase. Therefore, we determined the susceptibility of target DNA to exogenous DNase I after cytolysis by various means. We found no difference in DNA susceptibility for cells lysed by CTL vs methods (such as complement-mediated lysis or nonionic detergent) incapable of inducing nuclear disintegration. As a positive control, freezing and thawing dramatically enhanced susceptibility of the DNA. In conclusion, we found no evidence that the nuclear envelope is damaged by CTL in target cell types (or in the subpopulation of nuclei) that do not undergo nuclear disintegration.

Intracellular reovirus survives cytotoxic T lymphocyte-mediated lysis of its host cell.

Cytotoxic T lymphocytes (CTLs) induce rapid, extensive internal disintegration in target cells and this is unique among immune lytic mechanisms studied. This raises the question of whether CTLs are uniquely capable of halting virus infections by inducing damage within the target cell causing inactivation of intracellular virus. Reovirus infection of mouse P815 cells provided a suitable system for evaluating this question. An increase in cell-associated infectious virions began 8 h after infection and increased until 20 h post-infection, at which time the titre levelled off at about 100- to 1000-fold higher than the initial value. The infectious activity was compared between host cells killed by CTLs and those killed by sonication at various points in the infection cycle. The presence of reovirus within the target cell did not inhibit the usual internal disintegration events associated with the death of a target killed by CTLs. Nevertheless, the results indicated that CTLs were incapable of inactivating intracellular reovirus at any point in the life cycle of the virus: CTL-induced cytolysis simply released the infectious virions into the medium. Thus, at least in the case of reovirus, the utility of direct killing by CTLs would appear to be limited to reduction of the virus yield by lysis of the host cell before virus replication and assembly is completed.

The degree of CTL-induced DNA solubilization is not determined by the human vs mouse origin of the target cell.

CTL-mediated lysis is unique among lytic mechanisms in inducing rapid, prelytic nuclear disintegration. Target cell DNA can be solubilized within minutes as a result of degradation, which can proceed to the nucleosomal level, presumably mediated by endonucleases that are either endogenous or injected by the CTL. Nuclear disintegration has been reported for mouse lymphoid target cells by several groups. However, previous studies in which human target cells were studied saw little or no DNA solubilization. We here report rapid, extensive CTL-induced solubilization of DNA in human lymphoid target cells; on the other hand, we found that three mouse cell lines exhibit little or no nuclear disintegration. We conclude that the degree of nuclear disintegration depends on the nature of the target cell, but is not determined by the species of origin of the target cell.

Congestive heart failure due to giant cutaneous cavernous hemangioma.

A female infant is presented with isolated giant cutaneous cavernous hemangioma with secondary severe congestive heart failure. Studies to identify other major arteriovenous malformations were negative. An attempt to treat the patient with a corticosteroid was not successful in reducing the size of the hemangioma. She required an aggressive anticongestive medical regimen for 2 years. Though not previously described, high output congestive heart failure can occur secondary to isolated cutaneous hemangioma. Aggressive medical management may alleviate the need for the increased risk of surgical or other therapeutic approaches in this often self-limited condition.

Double aortic arch with patent ductus arteriosus in a premature infant: an unusual vascular malformation.

We recently encountered a premature infant with a very unusual form of vascular ring consisting of a double aortic arch with atretic left segment and patent ductus arteriosus. The initial presentation was that of patent ductus arteriosus with left-to-right shunting, but conventional surgical therapy was complicated by the abnormal aortic arch anatomy.

Phenotypic variation in biotinidase deficiency.

Biotinidase deficiency is the usual biochemical defect in biotin-responsive late-onset multiple carboxylase deficiency. We reviewed the clinical features of six patients with the enzyme deficiency and compared them with features described in the literature in children with late-onset MCD. In all of the reported probands, MCD was diagnosed because they had metabolic ketoacidosis and organic aciduria in addition to various neurologic and cutaneous symptoms, such as seizures, ataxia, skin rash, and alopecia. Although in several of our patients biotinidase deficiency was also diagnosed because they manifested a similar spectrum of findings, others never had ketoacidosis or organic aciduria. Thus the initial features of biotinidase deficiency usually include neurologic or cutaneous symptoms, whereas organic aciduria and MCD are delayed, secondary manifestations of the disease. These findings suggest that biotinidase deficiency should be considered in any infant or child with any of these neurologic or cutaneous findings, with or without ketoacidosis or organic aciduria. If the diagnosis cannot be excluded, such individuals should be given a therapeutic trial of pharmacologic doses of biotin.