Development and validation of a sleep questionnaire, SNoRE 3.0, to evaluate sleep in companion dogs.

Disturbances in the sleep-wake cycle are a debilitating, yet rather common condition not only in humans, but also in family dogs. While there is an emerging need for easy-to-use tools to document sleep alterations (in order to ultimately treat and/or prevent them), the veterinary tools which yield objective data (e.g. polysomnography, activity monitors) are both labor intensive and expensive. In this study, we developed a modified version of a previously used sleep questionnaire (SNoRE) and determined criterion validity in companion dogs against polysomnography and physical activity monitors (PAMs). Since a negative correlation between sleep time and cognitive performance in senior dogs has been demonstrated, we evaluated the correlation between the SNoRE scores and the Canine Dementia Scale (CADES, which includes a factor concerning sleep). There was a significant correlation between SNoRE 3.0 questionnaire scores and polysomnography data (latency to NREM sleep, ρ = 0.507, p < 0.001) as well as PAMs' data (activity between 1:00 and 3:00 AM, p < 0.05). There was a moderate positive correlation between the SNoRE 3.0 scores and the CADES scores (ρ = 0.625, p < 0.001). Additionally, the questionnaire structure was validated by a confirmatory factor analysis, and it also showed an adequate test-retest reliability. In conclusion the present paper describes a valid and reliable questionnaire tool, that can be used as a cost-effective way to monitor dog sleep in clinical settings.

T cell therapy against cancer: A predictive diffuse-interface mathematical model informed by pre-clinical studies.

T cell therapy has become a new therapeutic opportunity against solid cancers. Predicting T cell behaviour and efficacy would help therapy optimization and clinical implementation. In this work, we model responsiveness of mouse prostate adenocarcinoma to T cell-based therapies. The mathematical model is based on a Cahn-Hilliard diffuse interface description of the tumour, coupled with Keller-Segel type equations describing immune components dynamics. The model is fed by pre-clinical magnetic resonance imaging data describing anatomical features of prostate adenocarcinoma developed in the context of the Transgenic Adenocarcinoma of the Mouse Prostate model. We perform computational simulations based on the finite element method to describe tumor growth dynamics in relation to local T cells concentrations. We report that when we include in the model the possibility to activate tumor-associated vessels and by that increase the number of T cells within the tumor mass, the model predicts higher therapeutic effects (tumor regression) shortly after therapy administration. The simulated results are found in agreement with reported experimental data. Thus, this diffuse-interface mathematical model well predicts T cell behavior in vivo and represents a proof-of-concept for the role such predictive strategies may play in optimization of immunotherapy against cancer.

CD28 signaling augments Elk-1-dependent transcription at the c-fos gene during antigen stimulation.

Untransformed CD4(+) Th1 cells stimulated with Ag and APC demonstrated a dependence on B7- and CD28-mediated costimulatory signals for the expression and function of AP-1 proteins. The induction of transactivation by the c-fos gene regulator Elk-1 mirrored this requirement for TCR and CD28 signal integration. c-Jun N-terminal kinase (JNK) (but not extracellular signal-regulated kinase or p38) protein kinase activity was similarly inhibited by neutralizing anti-B7 mAbs. Blockade of JNK protein kinase activity with SB 202190 prevented both Elk-1 transactivation and c-Fos induction. These results identify a unique role for B7 costimulatory molecules and CD28 in the activation of JNK during Ag stimulation in Th1 cells, and suggest that JNK regulates Elk-1 transactivation at the c-fos gene to promote the formation of AP-1 complexes important to IL-2 gene expression.

Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells.

The effects of inflammatory cytokines on naive T cells have been studied using MHC protein/peptide complexes on microspheres, thus avoiding the use of APCs whose functions may be affected by the cytokines. IL-1, but not IL-12, increased proliferation of CD4+ T cells in response to Ag and IL-2, which is consistent with effects on in vivo priming of CD4+ cells. In contrast, proliferation of CD8+ T cells to Ag and IL-2 required IL-12, and IL-12 replaced adjuvant in stimulating an in vivo response to peptide. These results support a model in which distinct inflammatory cytokines act directly on naive CD4+ and CD8+ T cells to provide a third signal, along with Ag and IL-2, to optimally activate differentiation and clonal expansion.

A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles.

By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody (mAb) that recognizes the region around amino acid residue 44 on a non-native form of the protein. The conformational change in cyt c is an early event in apoptosis, which can be identified in pre-apoptotic cells that are negative for other indicators of apoptosis. Since the mAb did not bind fixed and permeabilized live cells and did not immunoprecipitate soluble cyt c extracted with detergent from dead cells, it appears to recognize cyt cbound in a detergent-sensitive complex to other cellular components. Coincidentally, the mAb was also shown by competitive enzyme-linked immunosorbent assay to bind cyt c associated with synthetic phosphatidic acid vesicles. This suggests that the conformational change of cyt c in dying cells could be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent confocal microscopy, conformationally altered cyt c in post-apoptotic T hybridoma cells showed a punctate distribution, indicating that it remained associated with mitochondria. Furthermore, the heavy membrane fraction of post-apoptotic cells but not of live cells was functional in caspase activation. This suggests that membrane-bound cyt c is the relevant caspase coactivation factor in the T hybridoma cells.

Structure and function of the urokinase receptor.

The binding of the urokinase plasminogen activator (uPA) to its receptor (uPAR) regulates cell adhesion, surface proteolysis, chemotaxis and cell extravasation in a number of experimental systems. Recent evidences have suggested that uPAR can by itself mediate chemotaxis of human monocytes and cause profound changes in cytoskeletal organization indicating that this receptor has the properties of a cell-surface regulated chemokine. Indeed, it is likely that upon binding to uPA, uPAR undergoes a conformational change that uncovers a new epitope located in the linker region between domain 1 and 2 of the receptor and is endowed with a potent chemotactic activity. This conformational change can be mimicked in vitro by enzymatic processing of a recombinant receptor. We have shown that chymotrypsin cleaves uPAR between domain 1 and 2 in an area that can be also cleaved by uPA at high efficiency and generate a receptor that can mediate monocytes migration independently of uPA binding. This mechanism is pertussis-toxin sensitive and involves activation of tyrosine kinases and cytoskeletal reorganization events in vitro. These studies indicate that in addition to its receptor function, upon binding to uPA, uPAR becomes a pleiotropic ligand for other still to be identified surface molecules.

Antigen-specific CD4+ T cells that survive after the induction of peripheral tolerance possess an intrinsic lymphokine production defect.

Injection of soluble foreign antigen without an adjuvant induces a state of antigen-specific immunological unresponsiveness. We investigated the cellular mechanisms that underlie this form of peripheral tolerance by physically tracking a small population of ovalbumin (OVA) peptide/I-Ad-specific, CD4+ T cell receptor (TCR) transgenic T cells following adoptive transfer into normal recipients. Injection of OVA peptide in the absence of adjuvant caused the antigen-specific T cells to proliferate for a brief period after which most of the T cells disappeared. The remaining OVA-specific T cells had converted to a memory phenotype but were poorly responsive in vivo as evidenced by a failure to accumulate in the draining lymph nodes following immunization with OVA peptide in adjuvant. These surviving T cells possessed a long-lasting, but reversible, defect in Il-2 and TNF-alpha production and in vivo proliferation, but did not gain capacity to produce Th2-type cytokines or suppress the clonal expansion of T cells specific for another antigen. Therefore, some antigen-specific T cells survive this peripheral tolerance protocol but are functionally unresponsive due to an intrinsic activation defect.

Direct evidence that functionally impaired CD4+ T cells persist in vivo following induction of peripheral tolerance.

A small population of CD4+ OVA-specific TCR transgenic T cells was tracked following the induction of peripheral tolerance by soluble Ag to address whether functionally unresponsive, or anergic T cells, persist in vivo for extended periods of time. Although injection of OVA peptide in the absence of adjuvant caused a transient expansion and deletion of the Ag-specific T cells, a population that showed signs of prior activation persisted in the lymphoid tissues for several months. These surviving OVA-specific T cells had long-lasting, but reversible defects in their ability to proliferate in lymph nodes and secrete IL-2 and TNF-alpha in vivo following an antigenic challenge. These defects were not associated with the production of Th2-type cytokines or the capacity to suppress the clonal expansion of a bystander population of T cells present in the same lymph nodes. Therefore, our results provide direct evidence that a long-lived population of functionally impaired Ag-specific CD4+ T cells is generated in vivo after exposure to soluble Ag.

A natural immunological adjuvant enhances T cell clonal expansion through a CD28-dependent, interleukin (IL)-2-independent mechanism.

The adoptive transfer of naive CD4(+) T cell receptor (TCR) transgenic T cells was used to investigate the mechanisms by which the adjuvant lipopolysaccharide (LPS) enhance T cell clonal expansion in vivo. Subcutaneous administration of soluble antigen (Ag) resulted in rapid and transient accumulation of the Ag-specific T cells in the draining lymph nodes (LNs), which was preceded by the production of interleukin (IL)-2. CD28-deficient, Ag-specific T cells produced only small amounts of IL-2 in response to soluble Ag and did not accumulate in the LN to the same extent as wild-type T cells. Injection of Ag and LPS, a natural immunological adjuvant, enhanced IL-2 production and LN accumulation of wild-type, Ag-specific T cells but had no significant effect on CD28-deficient, Ag-specific T cells. Therefore, CD28 is critical for Ag-driven IL-2 production and T cell proliferation in vivo, and is essential for the LPS-mediated enhancement of these events. However, enhancement of IL-2 production could not explain the LPS-dependent increase of T cell accumulation because IL-2-deficient, Ag-specific T cells accumulated to a greater extent in the LN than wild-type T cells in response to Ag plus LPS. These results indicate that adjuvants improve T cell proliferation in vivo via a CD28-dependent signal that can operate in the absence of IL-2.

Inflammatory cytokines enhance the in vivo clonal expansion and differentiation of antigen-activated CD4+ T cells.

Despite the wealth of information on the signals required for T cell activation in vitro, the signals required for the generation of functional Th cells in vivo are poorly understood. We addressed this by directly tracking the behavior of adoptively transferred CD4+ TCR transgenic T cells following Ag administration in vivo. Injection of soluble Ag induced a transient accumulation of Ag-specific T cells in lymphoid tissue. If bacterial LPS was present during this period, enhanced numbers of Ag-specific T cells accumulated, migrated into B cell-rich follicles, and provided help for Ab production. The ability of LPS to enhance the accumulation and follicular migration of Ag-activated T cells was mimicked by the proinflammatory cytokines, TNF-alpha and IL-1, and the capacity of LPS to promote the generation of IFN-gamma-secreting T cells, which provide help for IgG2a production, was mimicked by IL-12. Thus, the in vivo generation of functional Th cells can arise from Ag-dependent clonal expansion in the context of inflammatory cytokines.

In vivo detection of dendritic cell antigen presentation to CD4(+) T cells.

Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4(+) T cells specific for an OVA peptide-I-Ad complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell-rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.

Use of adoptive transfer of T-cell-antigen-receptor-transgenic T cell for the study of T-cell activation in vivo.

Adoptive transfer of TCR-transgenic T cells uniformly expressing an identifiable TCR of known peptide/MHC specificity can be used to monitor the in vivo behavior of antigen-specific T cells. We have used this system to show that naive T cells are initially activated within the T-cell zones of secondary lymphoid tissue to proliferate in a B7-dependent manner. If adjuvants or inflammatory cytokines are present during this period, enhanced numbers of T cells accumulate, migrate into B-cell-rich follicles, and acquire the capacity to produce IFN-gamma and help B cells produce IgG2a. If inflammation is absent, most of the initially activated antigen-specific T cells disappear without entering the follicles, and the survivors are poor producers of IL-2 and IFN-gamma. Our results indicate that inflammatory mediators play a key role in regulating the anatomic location, clonal expansion, survival and lymphokine production potential of antigen-stimulated T cells in vivo.

Defective transcription of the IL-2 gene is associated with impaired expression of c-Fos, FosB, and JunB in anergic T helper 1 cells.

Anergic CD4+ Th cells do not produce IL-2 when challenged with Ag-pulsed accessory cells because of a transcriptional defect. In this work, we report that these anergic T cells are defective in their ability to up-regulate protein binding and transactivation at two critical IL-2 DNA enhancer elements: NF-AT (nuclear factor of activated T cells; a sequence that binds a heterotrimeric NFATp, Fos, and Jun protein complex) and Activator Protein-1 (AP-1) (that binds Fos and Jun heterodimers). Western blot analysis of nuclear extracts showed that the impaired DNA-protein interactions in anergic T cells were associated with poor expression of the inducible AP-1 family members c-Fos, FosB, and JunB. However, the reduced expression of these proteins was not the result of a global TCR/CD3-signaling defect because CD3 cross-linking induced an equivalent increase in intracellular-free calcium ions, as well as NFATp dephosphorylation, translocation to the nucleus, and DNA binding in both normal and anergic T cells. Thus, defective IL-2 gene transcription appears to be due, at least in part, to a selective block in the expression of the AP-1 Fos and Jun family members in anergic T cells.

The anatomy of T-cell activation and tolerance.

The mammalian immune system must specifically recognize and eliminate foreign invaders but refrain from damaging the host. This task is accomplished in part by the production of a large number of T lymphocytes, each bearing a different antigen receptor to match the enormous variety of antigens present in the microbial world. However, because antigen receptor diversity is generated by a random mechanism, the immune system must tolerate the function of T lymphocytes that by chance express a self-reactive antigen receptor. Therefore, during early development, T cells that are specific for antigens expressed in the thymus are physically deleted. The population of T cells that leaves the thymus and seeds the secondary lymphoid organs contains helpful cells that are specific for antigens from microbes but also potentially dangerous T cells that are specific for innocuous extrathymic self antigens. The outcome of an encounter by a peripheral T cell with these two types of antigens is to a great extent determined by the inability of naive T cells to enter nonlymphoid tissues or to be productively activated in the absence of inflammation.

Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells.

T cells activated by antigen receptor stimulation in the absence of accessory cell-derived costimulatory signals lose the capacity to synthesize the growth factor interleukin-2 (IL-2), a state called clonal anergy. An analysis of CD3- and CD28-induced signal transduction revealed reduced ERK and JNK enzyme activities in murine anergic T cells. The amounts of ERK and JNK proteins were unchanged, and the kinases could be fully activated in the presence of phorbol 12-myristate 13-acetate. Dephosphorylation of the calcineurin substrate NFATp (preexisting nuclear factor of activated T cells) also remained inducible. These results suggest that a specific block in the activation of ERK and JNK contributes to defective IL-2 production in clonal anergy.

Accumulation of sequence-specific RNA-binding proteins in the cytosol of activated T cells undergoing RNA degradation and apoptosis.

Engagement of the T cell antigen receptor (TCR) causes transformed T cell hybridomas to produce lymphokines and then die by an apoptotic mechanism. Here we show that these functional effects of TCR-mediated signaling are associated with the accumulation of several cytosolic mRNA-binding proteins including the previously described AU-A, -B, and -C proteins as well as a novel 70-kDa protein. The results indicate that the 70-kDa protein derived from a 90-kDa precursor present in unactivated T cells and that both proteins bound to two independent sites at the 3'-end of the interleukin-2 mRNA, one in the coding region and the other in an AU-rich segment of the 3'-untranslated region. Glucocorticoids, TCR engagement by monoclonal antibodies, pharmacologic mimics of TCR signaling, or high concentrations of protein or RNA synthesis inhibitors all induced apoptosis, the cytosolic appearance of the RNA-binding proteins, and an increased rate of RNA turnover. Moreover, drugs that interfere with TCR-mediated signals, such as cyclosporin A and staurosporin, prevented both apoptosis and the appearance of the RNA-binding factors. The fact that the accumulation of these factors occurred in the presence of inhibitors of transcription and translation suggested that these proteins are present in an inactive form in unstimulated T cells and are activated when apoptosis is induced.

Prevalencia de bocio en escolares (6 a 14 años) de la ciudad de Mar del Plata / Goiter prevalence in school children (6 to 14 years) of Mar del Plata city

El objetivo de este trabajo fue determinar la prevalencia de bocio en escolares de la ciudad de Mar del Plata. Se seleccionó una muestra entre alumnos de 6 a 14 años de edad de escuelas urbanas y periurbanas de la ciudad de Mar del Plata; la muestra se realizó por conglomerados y posteriormente por métodos aleatorios. Se encuestaron 889 alumnos, 54,1 por ciento de sexo masculino y 45,9 por ciento del femenino, y se recolectó orina en un 8,7 por ciento de los varones. Se detectó un 17,2 por ciento del nivel Ia, 1,7 por ciento Ib y 0,1 por ciento II Total = 19 por ciento (según clasificación OPS). No se observaron diferencias significativas respecto al sexo y edad, ni a la provisión de agua corriente y/o de pozo ni a residencia urbana o rural. Si se observó diferencia significativa entre escuelas Estatales y Privadas : 21,2 por ciento y 11,6 por ciento respectivamente. Dado las diferencias observadas entre los distintos equipos de trabajo (todos los profesionales intervinientes con experiencia en palpación de tiroides), se resolvió efectuar ecografía a un 20 por ciento de los positivos más un 10 por ciento de normales : los resultados de las ecografías avalan ampliamente los hallazgos palpatorios. De las 53 muestras de orina procesadas, se observó un promedio de 21,1 mcg/dl de iodo con un desvío standar de 6,7 mcg/dl (es decir, iodurias altamente satisfactorias). Se ha decidido profundizar la investigación, determinando anticuerpos TPO y medición TSH, para destacar tiroiditis crónica e hipotiroidismo subclínico

Prevalencia de bocio en escolares (6 a 14 años) de la ciudad de Mar del Plata / Goiter prevalence in school children (6 to 14 years) of Mar del Plata city

El objetivo de este trabajo fue determinar la prevalencia de bocio en escolares de la ciudad de Mar del Plata. Se seleccionó una muestra entre alumnos de 6 a 14 años de edad de escuelas urbanas y periurbanas de la ciudad de Mar del Plata; la muestra se realizó por conglomerados y posteriormente por métodos aleatorios. Se encuestaron 889 alumnos, 54,1 por ciento de sexo masculino y 45,9 por ciento del femenino, y se recolectó orina en un 8,7 por ciento de los varones. Se detectó un 17,2 por ciento del nivel Ia, 1,7 por ciento Ib y 0,1 por ciento II Total = 19 por ciento (según clasificación OPS). No se observaron diferencias significativas respecto al sexo y edad, ni a la provisión de agua corriente y/o de pozo ni a residencia urbana o rural. Si se observó diferencia significativa entre escuelas Estatales y Privadas : 21,2 por ciento y 11,6 por ciento respectivamente. Dado las diferencias observadas entre los distintos equipos de trabajo (todos los profesionales intervinientes con experiencia en palpación de tiroides), se resolvió efectuar ecografía a un 20 por ciento de los positivos más un 10 por ciento de normales : los resultados de las ecografías avalan ampliamente los hallazgos palpatorios. De las 53 muestras de orina procesadas, se observó un promedio de 21,1 mcg/dl de iodo con un desvío standar de 6,7 mcg/dl (es decir, iodurias altamente satisfactorias). Se ha decidido profundizar la investigación, determinando anticuerpos TPO y medición TSH, para destacar tiroiditis crónica e hipotiroidismo subclínico

Surface proteins involved in T cell costimulation.

The activation and eventual clonal expansion of individual antigen-specific CD4+ T cell clones are dependent on the production of autocrine growth factors such as interleukin-2 (IL-2). The specificity of CD4+ T cell activation is imparted by T cell antigen receptor (TCR) recognition of peptide antigens bound to class II major histocompatibility complex (MHC)-encoded molecules on the surface of antigen-presenting cells (APCs), for example B cells, macrophages, and dendritic cells. To induce maximal IL-2 production by T cells, however, APCs must also provide non-antigen-specific costimulatory signals. Recent work indicates that APC-derived costimulatory signals play a critical role in determining whether lymphokine production, apoptotic cell death, or functional anergy is induced by TCR engagement. This information has allowed immunologists to manipulate costimulatory molecules to prevent allograft rejection and enhance tumor immunity. Here we review current information on the biologic effects of, and signal transduction pathways engaged by, several known receptor-ligand pairs that transduce costimulatory signals in T cells. Special emphasis will be placed on the interaction of CD28 on the T cell with its ligands, B7-1, B7-2, and B7-3 on the APC.