Infectious Bronchitis Virus Immune Responses in the Harderian Gland upon Initial Vaccination.

In recent years, Arkansas Delmarva Poultry Industry (ArkDPI)-derived infectious bronchitis (IB) virus (IBV) vaccines have been used to characterize the immune responses of chickens subsequent to vaccination on day of hatch or beyond. Perhaps because ArkDPI vaccines display increased heterogeneity, the results on cell immune responses have shown ambiguity. In the current study, we investigated the effects of vaccination with a highly stable and homogeneous Massachusetts (Mass)-type vaccine on days 1 or 7 of age on Harderian gland (HG) responses. Confirming previous studies, both IBV serum antibodies and lachrymal IgA levels were greater upon vaccination on day 7 compared with vaccination on day 1 of age. Unlike results with ArkDPI viruses, a clear trend was detected for both B and T cells in the HG after Mass-type vaccination. Consistent with antibody responses, B- and T-helper (CD3+CD4+) cell frequencies were higher in birds vaccinated on day 7 of age. Cytotoxic T cells (CD3+CD8+) were also increased compared with chickens vaccinated on day 1 of age. Depending on the most likely age of IB outbreaks to occur in a particular region, postponing the first IBV vaccination may optimize immune responses.

Nota de Investigación- Respuestas inmunes al virus de la bronquitis infecciosa en la glándula de Harder después de una vacunación inicial En los últimos años, las vacunas contra el virus de la bronquitis infecciosa (IB) derivadas de la cepa Arkansas Industria Avícola de Delmarva (ArkDPI) se han utilizado para caracterizar las respuestas inmunes de los pollos después de la vacunación en el día de la eclosión o en días posteriores. Posiblemente debido a que las vacunas ArkDPI muestran una mayor heterogeneidad, los resultados sobre las respuestas inmunes celulares han mostrado ambigüedad. En el presente estudio, se investigaron los efectos de la vacunación con una vacuna de tipo Massachusetts (Mass) que es altamente estable y homogénea en los días uno o siete de edad con relación a las respuestas en la glándula de Harder (HG). Confirmando estudios previos, tanto los anticuerpos séricos contra el virus de la bronquitis infecciosa como los niveles de IgA lacrimal fueron mayores tras la vacunación al día siete en comparación con la vacunación al primer día de edad. A diferencia de los resultados con los virus ArkDPI, se detectó una clara tendencia para las células B y T en la glándula de Harder después de la vacunación con el tipo Mass. De acuerdo con las respuestas de anticuerpos, las frecuencias de células B y T cooperadoras (CD3+ CD4+) fueron más altas en las aves vacunadas en el día siete de edad. Las células T citotóxicas (CD3+CD8+) también aumentaron en comparación con los pollos vacunados en el primer día de edad. Dependiendo de la edad más probable en que ocurran brotes por bronquitis infecciosa en una región en particular, posponer la primera vacuna contra el IBV puede optimizar las respuestas inmunes.

Early Vaccination of Chickens Induces Suboptimal Immunity Against Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) is highly prevalent in broiler chickens despite extensive vaccination commonly conducted early after hatch. The effects of early vaccination on immune responses were further investigated in chickens primed at increasing ages, followed by booster vaccination with an attenuated Arkansas (Ark) Delmarva Poultry Industry-type vaccine. Results show that vaccination on day 1 of age elicits significantly lower systemic and mucosal antibody responses compared with vaccination at later time points in the life of the chicken. The increase of IBV antibodies in serum from secondary responses after booster vaccination was more dramatic and significantly higher when measured by an Ark spike subunit 1 protein ELISA compared with measuring by non-Ark serotype whole-virus ELISA, which underlines the immunogenic importance of the virus spike at inducing antibodies. However, the levels achieved following boosting did not differ significantly between ages of priming. Thus, it seems that the booster vaccination mitigated the differences detected after prime immunization. In contrast to the continued rise of systemic antibodies after booster vaccination, the levels of mucosal IBV-specific immunoglobulin A decreased after booster vaccination. The recruitment or expansion of cluster of differentiation (CD)4+, CD8+, and CD4+/CD8+ T-cell populations in different immune effector sites was increased with age, but remained unaltered by IBV vaccination. In contrast, peripheral blood CD4+ cells showed a significant increase in IBV-vaccinated chickens compared with nonvaccinated age-matched controls both after primary and booster immunization. The results of the current study confirm that IBV vaccination on the day of hatch induces suboptimal IBV immune responses both in the systemic and mucosal compartments. This routine practice may be contributing to the immunologic escape of the virus and increased persistence of vaccine virus in vaccinated chickens. However, booster vaccination seems to overcome poor initial responses.

La vacunación temprana de pollos induce inmunidad subóptima contra el virus de la bronquitis infecciosa. El virus de la bronquitis infecciosa (IBV, por sus siglas en inglés) es altamente prevalente en los pollos de engorde a pesar de la vacunación extensiva que comúnmente se realiza temprano después de la eclosión. Los efectos de la vacunación temprana en las respuestas inmunitarias se investigaron más a fondo en pollos primovacunados a edades crecientes, seguidos de la vacunación de refuerzo con una vacuna atenuada tipo Arkansas (Ark) Delmarva Poultry Industry (Industria Avícola de Delmarva). Los resultados muestran que la vacunación en el día uno de edad provoca respuestas de anticuerpos sistémicos y de las mucosas significativamente menores en comparación con la vacunación en momentos posteriores en la vida del pollo. El aumento de los anticuerpos contra el virus de la bronquitis infecciosa en el suero en las respuestas secundarias después de la vacunación de refuerzo fue más marcado y significativamente mayor cuando se midió con un ensayo de inmunoabsorción con enzymas ligadas (ELISA) para la proteína de la subunidad 1 de la proteína de la espícula en comparación con la medición con un estuche de ELISA con el virus completo de serotipo diferente al Arkansas, lo que subraya la importancia inmunogénica de la espícula del virus en la inducción de anticuerpos. Sin embargo, los niveles alcanzados después del refuerzo no difirieron significativamente entre las edades de primovacunación. Por lo tanto, parece que la vacunación de refuerzo mitigó las diferencias detectadas después de la primo inmunización. En contraste con el aumento continuo de anticuerpos sistémicos después de la vacunación de refuerzo, los niveles de inmunoglobulina A específica para el virus de la bronquitis infecciosa en la mucosa disminuyeron después de la vacunación de refuerzo. El reclutamiento o la expansión de las poblaciones de células T CD4+, CD8+ y CD4+-CD8+ en diferentes sitios efectores inmunes se incrementó con la edad, sin embargo, permaneció inalterado por la vacunación contra el virus de la bronquitis infecciosa. En contraste, las células CD4+ de sangre periférica mostraron un aumento significativo en los pollos vacunados contra el virus de la bronquitis infecciosa en comparación con los controles no vacunados, tanto después de la primo inmunización como después de la inmunización de refuerzo. Los resultados de este estudio confirman que la vacunación contra el virus de la bronquitis infecciosa en el día de la eclosión induce respuestas inmunes subóptimas contra el mismo virus tanto en los compartimentos sistémicos como en los de las mucosas. Esta práctica de rutina puede contribuir al escape inmunológico del virus y al aumento de la persistencia del virus vacunal en pollos vacunados. Sin embargo, la vacunación de refuerzo parece mitigar las malas respuestas iniciales.

Infectious Bronchitis Virus Vaccination at Day 1 of Age Further Limits Cross Protection.

Cross-protection and immune responses elicited by infectious bronchitis virus (IBV) vaccination on Day 1 of age or at later time points were examined. Specific-pathogen-free chickens were vaccinated with a Massachusetts-type vaccine and heterologous challenge was performed with an Arkansas (Ark) -type virulent strain. In Trial 1, chickens vaccinated on Day 1 or Day 10 of age were challenged 21 days after vaccination. Analysis of tracheal histopathology and viral load demonstrated less cross protection when vaccination was performed on Day 1 of age. In Trial 2, chickens were vaccinated on Day 1 or Day 14 of age. A somewhat stronger systemic antibody response to IBV was detected in chickens vaccinated at 14 days of age. In addition, avidity of antibodies to Ark-type S1 protein elicited by vaccination at 14 days of age was greater. Differences in immune-cell populations in the Harderian gland (HG) observed at the time of sampling (35 days following vaccination) between chickens vaccinated at 1 day or 14 days of age indicated greater, rather than reduced, immune activity in the chickens vaccinated at 1 day of age. These differences are, perhaps, a result of the higher levels of persisting vaccine virus observed in the younger chickens. Both nonvaccinated/challenged groups showed significantly higher (P < 0.05) proportions of B cells and CD8+ T cells 7 days after challenge than age-matched vaccinated/challenged groups or age-matched nonvaccinated/nonchallenged control groups. These results indicate infiltration and/or expansion of B cells and CD8+ cells in HGs following challenge of nonvaccinated chickens. A fortuitous finding was that the more immature immune system of chickens vaccinated at 1 day of age was less effective at clearing vaccine virus after vaccination. Collectively, the current results indicate that IBV vaccination at 1 day of age can decrease the potential for heterologous cross protection compared with vaccination at least 10 days after hatch. A lower level of antibody affinity maturation likely contributes to decreased cross protection.

Límites en la protección cruzada inducida por la vacunación contra el virus de la bronquitis infecciosa en el primer día de edad. Se examinó la protección cruzada y la respuesta inmune inducida por la vacunación contra el virus de la bronquitis infecciosa en el primer día de edad o en posteriores puntos en el tiempo. Se vacunaron pollos libres de patógenos específicos con una vacuna de tipo Massachusetts y se realizó un desafío heterólogo con una cepa virulenta del tipo Arkansas (Ark). En el ensayo 1, pollos vacunados en el día uno o en el día 10 de edad fueron desafiados a los 21 días después de la vacunación. El análisis de la histopatología traqueal y de la carga viral demostró una menor protección cruzada cuando la vacunación se realizó en el día uno de edad. En el ensayo dos, los pollos fueron vacunados en el día uno o en el día 14 de edad. Se detectó una respuesta de anticuerpos sistémicos ligeramente más elevada contra el virus de la bronquitis infecciosa en los pollos vacunados a los 14 días de edad. Además, la avidez de los anticuerpos contra la proteína S1 tipo Arkansas inducida por la vacunación a los 14 días de edad fue mayor. Las diferencias en las poblaciones de células inmunitarias en la glándula de Harder observadas en el momento del muestreo 35 días después de la vacunación en los pollos vacunados al primer día o a los14 días de edad indicaron una mayor actividad inmunitaria en lugar de reducción en los pollos vacunados al primer día de edad, posiblemente como resultado de los niveles más altos de virus vacunal persistente observados en los pollos más jóvenes. Ambos grupos no vacunados y desafiados mostraron proporciones significativamente más altas (P <0.05) de células B y células T CD8+ siete días después del desafío en comparación con los grupos vacunados y desafiados de la misma edad o los grupos control no vacunados y no desafiados de la misma edad. Estos resultados indican la infiltración y/o expansión de las células B y de células CD8+ en la glándula de Harder después del desafío de los pollos no vacunados. Un hallazgo fortuito fue que el sistema inmune más inmaduro de los pollos vacunados al primer día de edad fue menos efectivo para eliminar el virus vacunal después de la vacunación. En conjunto, los resultados actuales indican que la vacunación con el virus de la bronquitis infecciosa al primer día de edad puede disminuir el potencial de protección cruzada heteróloga en comparación con la vacunación al menos 10 días después de la eclosión. Un nivel más bajo de maduración de la afinidad de anticuerpos probablemente contribuye a disminuir la protección cruzada.

Conjunctiva-associated lymphoid tissue in avian mucosal immunity.

Conjunctiva-associated lymphoid tissue's (CALT) role in generating avian mucosal adaptive immunity was measured by analyzing cellular composition, expression of the polymeric immunoglobulin receptor (pIgR), and production of cytokines and antibodies in chickens ocular exposed to a replication-deficient adenovirus of serotype 5 (Ad5). These studies demonstrate that CALT contains B cells, γδ T cells, T helper, and cytotoxic T cells, and a T lymphocyte composition, which more resembles Harderian glands than spleen. CALT-derived lymphocytes contain antigen-specific, IgA-secreting plasma cells and cytokine-producing lymphocytes after ocular Ad5 vaccination. The expression of the pIgR in the CALT's lymphoepithelium emphasizes the importance of mucosal immune protection by paraocular lymphoid tissues. The CALT immune response after ocular Ad5 boosting was influenced by prior high dose in ovo Ad5 priming. Thus, both mucosal and systemic immunization influenced Ad5-induced IFN-γ responses in CALT.

Infectious bronchitis virus in the chicken Harderian gland and lachrymal fluid: viral load, infectivity, immune cell responses, and effects of viral immunodeficiency.

We compared detection of infectious bronchitis virus (IBV) by quantitative RT-PCR (qRT-PCR) in tears and trachea of IBV-infected chickens and found that quantitative detection of IBV RNA in tears is more sensitive than in tracheal homogenates. Furthermore, we demonstrated that IBV contained in chicken lachrymal fluid is infectious and that tears of IBV-infected chickens can be used to infect naive chickens. We compared the immune responses to IBV in the Harderian gland and cecal tonsils of immunocompetent chickens and chickens infected with chicken anemia virus (CAV) and/or infectious bursal disease virus (IBDV). Flow cytometry analyses of lymphocytes in Harderian glands and cecal tonsils indicated that the relative abundance of IgM+ B cells in the Harderian glands and cecal tonsils following exposure to IBV in combination with immunosuppressive viruses was reduced compared to chickens infected with IBV alone. CAV, but not IBDV, reduced the CD4+/CD8+ T cell ratios compared to chickens infected with IBV alone. Enzyme-linked immuno-spot forming assays on cells in the Harderian glands and cecal tonsils of IBV-infected chickens indicated that maximum IBV-specific IgA-secreting cell responses were reduced in chickens infected with CAV. IBDV co-infected chickens displayed a delayed IgA response to IBV. Thus immunosuppressive viruses reduced B cells and T helper cells in the Harderian glands and cecal tonsils in response to IBV, and slowed the kinetics and/or reduced the magnitude of the mucosal immune response against IBV. We have shown for the first time that CAV affects pathogen-specific B cell responses in a mucosal effector site.

The PTPmu protein-tyrosine phosphatase binds and recruits the scaffolding protein RACK1 to cell-cell contacts.

PTPmu, an Ig superfamily receptor protein-tyrosine phosphatase, promotes cell-cell adhesion and interacts with the cadherin-catenin complex. The signaling pathway downstream of PTPmu is unknown; therefore, we used a yeast two-hybrid screen to identify additional PTPmu interacting proteins. The membrane-proximal catalytic domain of PTPmu was used as bait. Sequencing of two positive clones identified the scaffolding protein RACK1 (receptor for activated protein C kinase) as a PTPmu interacting protein. We demonstrate that RACK1 interacts with PTPmu when co-expressed in a recombinant baculovirus expression system. RACK1 is known to bind to the src protein-tyrosine kinase. This study demonstrates that PTPmu association with RACK1 is disrupted by the presence of constituitively active src. RACK1 is thought to be a scaffolding protein that recruits proteins to the plasma membrane via an unknown mechanism. We have shown that the association of endogenous PTPmu and RACK1 in a lung cell line is increased at high cell density. We also demonstrate that the recruitment of RACK1 to both the plasma membrane and cell-cell contact sites is dependent upon the presence of the PTP mu protein in these cells. Therefore, PTPmu may be one of the proteins that recruits RACK1 to points of cell-cell contact, which may be important for PTPmu-dependent signaling in response to cell-cell adhesion.

PTPmu regulates N-cadherin-dependent neurite outgrowth.

Cell adhesion is critical to the establishment of proper connections in the nervous system. Some receptor-type protein tyrosine phosphatases (RPTPs) have adhesion molecule-like extracellular segments with intracellular tyrosine phosphatase domains that may transduce signals in response to adhesion. PTPmu is a RPTP that mediates cell aggregation and is expressed at high levels in the nervous system. In this study, we demonstrate that PTPmu promotes neurite outgrowth of retinal ganglion cells when used as a culture substrate. In addition, PTPmu was found in a complex with N-cadherin in retinal cells. To determine the physiological significance of the association between PTPmu and N-cadherin, the expression level and enzymatic activity of PTPmu were perturbed in retinal explant cultures. Downregulation of PTPmu expression through antisense techniques resulted in a significant decrease in neurite outgrowth on an N-cadherin substrate, whereas there was no effect on laminin or L1-dependent neurite outgrowth. The overexpression of a catalytically inactive form of PTPmu significantly decreased neurite outgrowth on N-cadherin. These data indicate that PTPmu specifically regulates signals required for neurites to extend on an N-cadherin substrate, implicating reversible tyrosine phosphorylation in the control of N-cadherin function. Together, these results suggest that PTPmu plays a dual role in the regulation of neurite outgrowth.

Dealing with the dilemmas of confidentiality.

The duty of nurses to maintain patient confidentiality can be influenced by a number of factors. This article discusses the circumstances under which nurses may decide, or be constrained, to share information about a patient. It argues for greater clarity in the guidance given to nurses regarding confidentiality.

The role of cell adhesion molecule L1 in axonal extension, growth cone motility, and signal transduction.

Axonal pathfinding is a complex process dependent on cell-cell and cell-matrix interactions. L1 is a cell adhesion molecule that is abundant in the nervous system and that is concentrated on axons. As a culture substrate, L1 is a potent promoter of neurite outgrowth and elicits specific growth cone behavior. It interacts with the actin cytoskeleton via an ankyrin linkage and promotes specific distribution of F-actin within the growth cone. In addition, L1 has been implicated in signal transduction. For example, L1 is associated with kinases, L1-L1 binding regulates second messenger systems, and mutations in the L1 gene in humans result in abnormalities in the development of the corticospinal tract and corpus callosum. In this short review, recent advances in understanding the way in which L1 regulates growth cone behavior will be discussed.

L1, N-cadherin, and laminin induce distinct distribution patterns of cytoskeletal elements in growth cones.

Growth cones are highly motile extensions of growing neuronal processes that have a well-characterized cytoskeleton, which is necessary for motility and directed growth. In culture, neurons have been observed to extend processes on a variety of substrates made of cell adhesion molecules (CAMs) or extracellular matrix molecules (ECM molecules). We have previously shown that retinal ganglion cell (RGC) growth cones exhibit characteristic morphologies dependent on the substrate on which they are grown (Payne et al., 1992: Cell Motil. Cytoskel. 21:65-73). Upon contact with a sharp border between two substrates, the growth cones display rapid changes in morphology (Burden-Gulley et al., 1995: J. Neurosci. 15:4370-4381) that may result from extensive restructuring of the cytoskeleton. In the present study, immunocytochemical methods were used to examine the distribution of three cytoskeletal elements in RGC growth cones growing on L1, N-cadherin, or laminin as well as on dishes coated with alternating lanes of these substrates. Distinct distribution patterns of f-actin, microtubules (MTs), and neurofilaments (NFs) were observed in growth cones growing on individual substrates. At border regions between two substrates, growth cones with extensive lamellipodial contact with the second substrate were observed to have f-actin and MT distribution patterns appropriate for the new substrate encountered. Contact via filopodia alone did not evoke this change. Redistribution of NFs was observed only after the majority of the growth cone had crossed onto the second substrate. These results suggest that actin and MTs, but probably not NFs, are directly influenced by CAMs and ECM molecules to produce changes in growth cone morphology. The distribution of two members of the protein tyrosine kinase family, pp60arc and p59fyn, and phosphorylated tyrosine residues was also examined. No differences were observed in the distribution patterns of the kinases and phosphorylated tyrosine residues in growth cones on any of the substrate molecules tested. In addition, the distribution patterns were unchanged in growth cones that contacted and crossed borders between two substrates. These results suggest that redistribution of pp60arc or p59fyn is not required to produce alterations in growth cone morphology induced by contact with L1, N-cadherin or laminin.

Growth cones are actively influenced by substrate-bound adhesion molecules.

As axons advance to appropriate target tissues during development, their growth cones encounter a variety of cell adhesion molecules (CAMs) and extracellular matrix molecules (ECM molecules). Purified CAMs and ECM molecules influence neurite outgrowth in vitro and are thought to have a similar function in vivo. For example, when retinal ganglion cell (RGC) neurons are grown on different CAM and ECM molecule substrates in vitro, their growth cones display distinctive morphologies (Payne et al., 1992). Similarly, RGC growth cones in vivo have distinctive shapes at different points in the pathway from the eye to the tectum, suggesting the presence of localized cues that determine growth cone behaviors such as pathway selection at choice points. In this report, time-lapse video microscopy was utilized to examine dynamic transformations of RGC growth cones as they progressed from L1/8D9, N-cadherin, or laminin onto a different substrate. Contact made by the leading edge of a growth cone with a new substrate resulted in a rapid and dramatic alteration in growth cone morphology. In some cases, the changes encompassed the entire growth cone including those regions not in direct contact with the new substrate. In addition, the growth cones displayed a variety of behavioral responses that were dependent upon the order of substrate contact. These studies demonstrate that growth cones are actively affected by the substrate, and suggest that abrupt changes in the molecular composition of the growth cone environment are influential during axonal pathfinding.