Using Patient-Reported Outcome Measures for Quality Improvement in Clinical Genetics: an Exploratory Study.

International advocacy of patient-centred healthcare delivery has led to emphasis on the (re)design and evaluation of healthcare processes and outcomes from a patient perspective. Patient-reported outcome measures (PROMs) have significant potential to inform such attempts. However there is limited understanding of the processes by which this can be achieved. This exploratory study followed attempts to utilise two different PROMs measures to support service quality improvement in clinical genetics. PROMs used were the Genetic Counseling Outcome Scale (GCOS-24), a well-validated clinical genetics-specific PROM and Euroqol (EQ-5D), a generic PROM favoured by the UK National Institute for Health and Excellence (NICE). Both of these PROMs enable pre/post intervention comparison. A service audit tool was also used, premised on a patient-reported experience measure. In addition, the study draws on interviews with clinical staff to identify challenges associated with the use of PROMs (response rate, data collection, analysis). Benefits are also explored and include the provision of insight into patients' needs; complementing clinical judgement; identification of needs being met, evidencing the benefit of services provided; prompting consideration of areas requiring attention; and encouraging professional development.

Stable, stoichiometric delivery of diverse protein functions.

As contemporary "genomics" steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.

Characterization of F21.A, a monoclonal antibody that recognize a leucocyte surface antigen for killer whale homologue to beta-2 integrin.

The specificity of F21.A, a monoclonal antibody raised against bottlenose dolphin leucocytes, was characterized in killer whale on the basis of immunoprecipitation of a protein of 94 kDa, as well as flow cytometric analysis. While minimally expressed on resting cells, F21.A labeled a homologue to beta-2 integrin in 89-97% of PMA-activated neutrophils, 53-66% of activated monocytes, and activated B cells but not T cells. Activation of neutrophils reached its maximum 10 min after PMA stimulation. F21.A did not label intracellular stores as did both cross-reacting anti-canine CD11b and CD18, suggesting that an activation-induced conformational change would expose a neoepitope recognized by F21.A. F21.A labeling was largely inhibited by pre-incubation with plasma, suggesting a binding site closely related to that for fibrinogen. In vitro phagocytosis and respiratory burst were almost fully inhibited upon pre-incubation with F21.A, demonstrating its functional importance. This antibody is foreseen as a possible valuable diagnostic and research tool in cetacean immunology.

Measurement of intracellular gamma-interferon, interleukin-4, and interleukin-10 levels in patients following laparoscopic cholecystectomy.

Major surgery suppresses intracellular T-cell cytokine production. Laparoscopic surgery has been reported to have no effect on in vitro lymphocyte reactivity, but its effects on intracellular cytokine production are unknown. This study measured T-cell intracellular gamma-interferon, interleukin-4 (IL-4), and interleukin-10 (IL-10), along with serum interleukin-6 (IL-6) and cortisol levels, immediately before and 1 day after laparoscopic cholecystectomy in a cohort of six Air Force and veteran patients. Stimulated intracellular levels of gamma-interferon were slightly, but not significantly, elevated during the postoperative period in all T-cell subsets. There were no postoperative changes in stimulated IL-4 or IL-10 levels. Postoperative serum IL-6 levels, but not serum cortisol levels, were significantly elevated compared to preoperative values. In conclusion, laparoscopic surgery causes slight trauma but has no effect on T-cell intracellular interferon, IL-4, and IL-10 responses.

Deficiency in the transcription factor interferon regulatory factor (IRF)-2 leads to severely compromised development of natural killer and T helper type 1 cells.

Interferon (IFN) regulatory factor (IRF)-2 was originally described as an antagonist of IRF-1-mediated transcriptional regulation of IFN-inducible genes. IRF-1(-/)- mice exhibit defective T helper type 1 (Th1) cell differentiation. We have used experimental leishmaniasis to show that, like IRF-1(-/)- mice, IRF-2(-/)- mice are susceptible to Leishmania major infection due to a defect in Th1 differentiation. Natural killer (NK) cell development is compromised in both IRF-1(-/)- and IRF-2(-/)- mice, but the underlying mechanism differs. NK (but not NK(+) T) cell numbers are decreased in IRF-2(-/)- mice, and the NK cells that are present are immature in phenotype. Therefore, like IRF-1, IRF-2 is required for normal generation of Th1 responses and for NK cell development in vivo. In this particular circumstance the absence of IRF-2 cannot be compensated for by the presence of IRF-1 alone. Mechanistically, IRF-2 may act as a functional agonist rather than antagonist of IRF-1 for some, but not all, IFN-stimulated regulatory element (ISRE)-responsive genes.

Retroviral delivery of peptide modulators of cellular functions.

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.

Role of T cells and cytokines in fatal and resolving experimental babesiosis: protection in TNFRp55-/- mice infected with the human Babesia WA1 parasite.

We characterized the cytokine response and T-cell requirements of mice infected with the intraerythrocytic parasites Babesia microti and WA1. WA1 infections were fatal, whereas B. microti infections were resolved. We measured production of tumor necrosis factor (TNF)-alpha, interferon-gamma, interleukin (IL)-10, and IL-4 by splenic CD4+, CD8+, and gammadelta+ T cells using flow cytometry. WA1 inoculation stimulated TNF-alpha production, whereas resolving B. microti infections were characterized by increased IL-10 and IL-4. The role of TNF-alpha in WA1 infections was further investigated by inoculating TNFRp55-/- mice with a lethal dose of WA1. A survival rate of 90% in the TNFRp55-/- mice indicated that a disruption in the TNF-alpha pathway abrogated the pathologic mechanism of WA1. Inoculation of WA1 into CD4-/- and CD8-/- mice resulted in survival rates of 60% and 78%, respectively, whereas WA1 infection in gammadelta-/- and control mice was fatal. These results suggest that CD8+ T cells may contribute to the WA1-associated disease. Babesia-infected CD4-/- mice experienced a longer duration of parasitemia, indicating that CD4+ T cells participate in parasite elimination. These studies demonstrate differences in immune responses during fatal or resolving Babesia infections, and they identify TNF-alpha as an important mediator of the WA1-associated pathogenesis.

Up-regulation of tumor necrosis factor-alpha and interferon-gamma expression in the spleen and lungs of mice infected with the human Babesia isolate WA1.

We analyzed cytokine expression in mice infected with the intraerythrocytic parasites Babesia microti and WA1. In C3H/HeN mice, WA1 infections were fatal, whereas B. microti infections were resolved. We propose that the proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) contribute to the WA1-associated disease. WA1 infection was characterized by up-regulation of TNFalpha and IFNgamma mRNA in the spleen. Previous studies in WA1-infected mice showed that pathologic lesions occurred primarily in the lungs, including pulmonary edema and intravascular margination of leukocytes. Analysis of cytokine expression in the lungs is important for an understanding of the disease process in WA1-infected mice. Expression of both TNFalpha and IFNgamma mRNA was increased in the lungs of WA1-infected mice. Immunohistochemical staining confirmed the upregulation of these proinflammatory cytokines in the lungs. Expression of TNFalpha and IFNgamma was not up-regulated in the lungs of B. microti-infected mice. The results implicate TNFalpha and IFNgamma in the pathogenesis of WA1-associated disease.

Association of SWAP-70 with the B cell antigen receptor complex.

SWAP-70 is a component of an enzyme complex that recombines Ig switch regions in vitro. We report here the cloning of the human cDNA and its B lymphocyte-specific expression. Although its sequence contains three nuclear localization signals, in small resting B cells, SWAP-70 is mainly found in the cytoplasm. On stimulation, SWAP-70 translocates to the nucleus. In activated, class-switching B cell cultures, it is associated with membrane IgG, but not IgM. The membrane Ig association requires a functional pleckstrin homology domain and is controlled by the C terminus. We suggest that SWAP-70 is involved not only in nuclear events but also in signaling in B cell activation.

Murine T cell determination of pregnancy outcome.

At the fetomaternal interface, maternal effector cells come in intimate contact with fetal trophoblast cells which express paternal antigens. Failure of fetal trophoblast cells to activate maternal Th1 immune responses has been attributed in part to the absence of classical Class I and Class II major histocompatibilty complex (MHC) antigen expression and elaboration of factors which reduce TcR expression and shift any immune responses which may occur to Th2. Classical TcR alphabeta(+) T cells have not been found to be able to respond to trophoblasts. Recently, TcR gammadelta(+) T cells have been characterized in the low-abortion-rate pregnant C57Bl/10 mouse decidua, and the Vgamma1(+) subset may be able to respond to trophoblasts in a non-MHC-dependent manner. Trophoblast-recognizing T cells with Vgamma1 receptors are also present in the decidua of CBA/J mice pregnant by DBA/2, an abortion-prone mating combination. To test the role of the Vgamma1 subset of decidual gammadelta T cells in abortion-prone pregnancies, we altered this subset by injecting monoclonal anti-Vgamma1.1 antibody on gestation day 5.5, 1 day after implantation. This reduced detectability of a Vgammadelta subset producing TNF-alpha and reduced the abortion rate. Anti-Vgamma2, which reacts with a similar proportion of decidual gammadelta T cells as anti-Vgamma1.1, failed to prevent abortions. Vdelta6.3(+) cells are prominent at the fetomaternal interface, and anti-Vdelta6 antibody injected on day 5.5 prevented abortions. TGF-beta2(+) gammadelta cells first appear on day 8.5 of pregnancy; anti-Vgamma1.1 antibody injection on day 8.5 depleted these cells and boosted abortions; anti-Vdelta6.3 given on day 8.5 boosted abortions to the same level. These results suggest that two populations of Vgamma1.1(+)delta6.3(+) T cells may arise in the decidua: an early population that is Th1, abortogenic, and present during the time of implantation, and a Th2/3 cell subset that is present in the decidua later during pregnancy and which is pregnancy-protective.

Cutting edge: protective response to pulmonary injury requires gamma delta T lymphocytes.

Gamma delta intraepithelial lymphocytes are thought to coordinate responses to pathogens that penetrate the epithelial barrier. To directly test this, mice were inoculated with Nocardia asteroides. At doses that were nonlethal for control mice, gamma delta-deficient mice became severely ill and died within 14 days. Histologic examination of these lungs demonstrated the presence of severe tissue damage and unimpeded bacterial growth in the gamma delta-deficient mice compared with neutrophilic lesions and clearance of the organism in control mice. Interestingly, ozone exposure that targets a comparable lung region also resulted in diffuse epithelial necrosis associated with a similar lack of neutrophil recruitment in gamma delta-deficient mice. These data demonstrate that gamma delta intraepithelial lymphocytes can protect the host from pathogenic and nonpathogenic insults by targeting the inflammatory response to epithelial necrosis.

Major surgery suppresses maximal production of helper T-cell type 1 cytokines without potentiating the release of helper T-cell type 2 cytokines.

BACKGROUND: Major surgery is known to suppress T-cell function; however, its differential effects on the production of helper T-cell type 1 (T(H)1) and type 2 (T(H)2) cytokines remains unknown. OBJECTIVE: To measure the production patterns of T(H)1 (interleukin 2 [IL-2] and interferon gamma) and T(H)2 (IL-4 and IL-10) cytokines following major surgery. DESIGN, SETTING, AND PATIENTS: A cohort study of patients (both active and former members of the armed forces) at a military hospital. INTERVENTION: Aortic surgery or carotid endarterectomy and measurement of serum IL-6 levels by enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Unstimulated and stimulated intracellular levels of IL-2, IL-4, IL-10, and interferon gamma in CD4+, CD8+, and gammadelta+ T cells and serum IL-6 levels immediately before and for 2 days after aortic surgery or carotid endarterectomy. RESULTS: No unstimulated production of T(H) or T(H)2 cytokines was detected. Stimulated intracellular levels of IL-2 and interferon gamma were significantly depressed during the postoperative period in all T-cell subsets in both patient groups. There were no postoperative increases in stimulated IL-4 or IL-10 levels. CONCLUSION: Major surgery suppresses the potential responses of T(H)1 cytokines without enhancing production of T(H)2 cytokines.

Antigenic and nucleotide characterization of a herpesvirus isolated from Pacific harbor seals (Phoca vitulina richardsii).

This report describes the antigenic and nucleotide characterization of a herpes-like virus that has been isolated from the adrenal tissues of neonatal Pacific harbor seals. Infection with this virus has been previously implicated as a major cause of death of animals undergoing rehabilitation. Comparison and phylogenetic analysis of sequenced fragments of the DNA polymerase, glycoprotein B and glycoprotein D genes, and immunofluorescence assay using herpesvirus-specific monoclonal antibodies, demonstrated close similarity of the Pacific harbor seal herpesvirus to European isolates of phocid herpesvirus-1 (PHV-1) and other alpha-herpesviruses affecting terrestrial carnivores.

Characterization of a monoclonal antibody that recognizes a lymphocyte surface antigen for the cetacean homologue to CD45R.

As part of our current efforts to develop assays and reagents to study the immune system of marine mammals, and in view of the effort currently made to develop monoclonal antibodies to cell surface proteins of lymphocyte subsets in different species, the present paper reports on the characterization of a monoclonal antibody against the homologue of CD45R on cetacean lymphocytes. The specificity of this antibody has been characterized on the basis of immunoprecipitation of the antigen it recognized, immunoperoxidase staining on cetacean lymph node and thymus sections, as well as one and two-colour flow cytometric analysis of cetacean peripheral blood mononuclear cells and single-cell suspensions of thymus, lymph node and spleen. Anticetacean CD45R (F21.H) immunoprecipitated proteins of 180, 200 and 220 x 10(3) MW, with the 180 x 10(3) MW from being predominantly expressed on T cells and the 220 x 10(3) MW form expressed predominantly on B cells and thymocytes F21.H labelled all B cells and a proportion of T cells on single-cell suspensions of spleen cells. CD45R- killer whale peripheral blood lymphocytes expressed a higher density of CD2 than CD45R+, a characteristic of memory T cells. Killer whale T lymphocytes also lost the expression of CD45R upon activation with concanavalin A (Con A) and phytohaemagglutinin (PHA). This is the first report of a monoclonal antibody to CD45R in cetaceans, and this antibody is foreseen as a possible valuable diagnostic and research tool to assess immune functions of captive and wild cetaceans as part of the evaluation of their health status.

Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4(+) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes.

Gastrointestinal complications in human immunodeficiency virus (HIV) infection are indicative of impaired intestinal mucosal immune system. We used simian immunodeficiency virus (SIV)-infected rhesus macaques as an animal model for HIV to determine pathogenic effects of SIV on intestinal T lymphocytes. Intestinal CD4(+) T-cell depletion and the potential for cytokine responses were examined during SIV infection and compared with results for lymphocytes from lymph nodes and blood. Flow cytometric analysis demonstrated severe depletion of CD4(+)CD8(-) single-positive T cells and CD4(+)CD8(+) double-positive T cells in intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) during primary SIV infection which persisted through the entire course of SIV infection. In contrast, CD4(+) T-cell depletion was gradual in peripheral lymph nodes and blood. Flow cytometric analysis of intracellular gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production following short-term mitogenic activation revealed that LPL retained same or higher capacity for IFN-gamma production in all stages of SIV infection compared to uninfected controls, whereas peripheral blood mononuclear cells displayed a gradual decline. The CD8(+) T cells were the major producers of IFN-gamma. There was no detectable change in the frequency of IL-4-producing cells in both LPL and peripheral blood mononuclear cells. Thus, severe depletion of CD4(+) LPL and IEL in primary SIV infection accompanied by altered cytokine responses may reflect altered T-cell homeostasis in intestinal mucosa. This could be a mechanism of SIV-associated enteropathy and viral pathogenesis. Dynamic changes in intestinal T lymphocytes were not adequately represented in peripheral lymph nodes or blood.

Adrenocortical response and regional T-lymphocyte activation patterns following minimally invasive surgery in a rat model.

BACKGROUND: Laparoscopic surgery is associated with less tissue trauma and postoperative pain as well as a more rapid recovery than open surgery. We hypothesized that these factors may result in less immune impairment following laparoscopic surgery. METHODS: We measured mitogen-induced surface interleukin-2 receptor (IL2R) expression and lymphocyte proliferation in CD4+ and CD8+ T-lymphocytes as well as serum corticosterone levels in rats 24 h following open (OP) and laparoscopic (LAP) fundoplication. RESULTS: Serum corticosterone levels were lower in LAP vs OP rats (p = 0.02). CD4+ IL2R expression was higher in the blood, but not in the spleen, in LAP vs OP animals (p = 0.02). CD8+ IL2R expression was similar in both groups. Mitogen-induced lymphocyte proliferation was no different in the blood but decreased in the spleen in LAP vs OP rats (p = 0.03). CONCLUSIONS: Compared to open surgery, laparoscopic fundoplication in the rat results in lower adrenocortical hormone levels and better-preserved T-helper-cell activation in the blood. Lymphocyte proliferation is suppressed in the spleen 24 h after laparoscopic surgery. Minimally invasive surgery may better preserve cell-mediated immunity in the early postoperative period.

The induction of a protective response in Leishmania major-infected BALB/c mice with anti-CD40 mAb.

A protective immune response to the intracellular parasite Leishmania major requires the development of a Th1 CD4+ T cell phenotype. We demonstrate herein that BALB/c mice, which normally develop a susceptible Th2 response to L. major infection, are protected when co-injected with an agonistic anti-murine CD40 mAb. Anti-CD40 mAb-mediated protection in this system was found to be T cell dependent, since it was not observed in C57BL/6 x 129 mice that were rendered T cell deficient (TCR beta-/- x TCR delta-/-) and L. major susceptible. Anti-CD40 mAb stimulation of L. major-infected BALB/c mice was accompanied by increased IL-12 and IFN-gamma production in draining lymph nodes, analyzed either by direct expression, or in an antigen-specific in vitro recall assay. The protective role of these cytokines was indicated by the finding that anti-CD40 mAb-mediated protection of L. major-infected BALB/c mice could be reversed by co-treating the animals with neutralizing anti-IL-12 and/or anti-IFN-gamma mAb. Collectively, these data suggest that BALB/c mice develop a protective Th1 CD4+ T cell response to L. major infection when co-injected with anti-CD40 mAb. While the CD40-CD40L interaction has been previously shown to be vital in the control of murine Leishmaniasis, the current study establishes in vivo that anti-CD40 mAb treatment alone is sufficient to protect BALB/c mice from L. major infection and raises the possibility of utilizing this approach for vaccination strategies.

Early lethality, functional NF-kappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice.

TRAF2 is an intracellular signal-transducing protein recruited to the TNFR1 and TNFR2 receptors following TNF stimulation. To investigate the physiological role of TRAF2, we generated TRAF2-deficient mice. traf2-/- mice appeared normal at birth but became progressively runted and died prematurely. Atrophy of the thymus and spleen and depletion of B cell precursors also were observed. Thymocytes and other hematopoietic progenitors were highly sensitive to TNF-induced cell death and serum TNF levels were elevated in these TRAF2-deficient animals. Examination of traf2-/- cells revealed a severe reduction in TNF-mediated JNK/SAPK activation but a mild effect on NF-kappaB activation. These results suggest that TRAF2-independent pathways of NF-kappaB activation exist and that TRAF2 is required for an NF-kappaB-independent signal that protects against TNF-induced apoptosis.