Itinerário da família para obter o diagnóstico da criança com necessidades especiais de saúde / Itinerario de la familia para obtener el diagnóstico del niño con necesidades especiales de salud / The itinerary of families to obtain the diagnosis of the children with special healthcare needs

RESUMO Objetivo: conhecer o itinerário percorrido pela família para a obtenção do diagnóstico da criança com necessidades especiais de saúde. Método: realizou-se estudo qualitativo em uma unidade de internação pediátrica do sul do Brasil. Utilizou-se como referencial metodológico a Grounded Theory. Participaram 16 familiares cuidadores. Os dados foram coletados em 2019 por entrevistas e submetidos à codificação aberta e axial. Resultados: as famílias percorreram um longo itinerário até o recebimento do diagnóstico da criança: detectaram alterações no seu estado geral, realizaram diversos exames, desconfiaram o diagnóstico por já ter outros com o mesmo na família, receberam informações dos profissionais da saúde acerca dos cuidados necessários e dos serviços especializados para o seu tratamento, apresentaram medo, pânico, negação e valorizaram a fé em Deus na esperança da melhora da criança. Considerações finais: o enfermeiro necessita desenvolver um processo educativo junto à família para que ela se sinta preparada e capaz de cuidar dessas crianças.

RESUMEN Objetivo: conocer el itinerario recorrido por la familia para la obtención del diagnóstico del niño con necesidades especiales de salud. Método: se realizó estudio cualitativo en una unidad de hospitalización pediátrica del sur de Brasil. Se utilizó como referencial metodológico la GroundedTheory. Participaron 16 familiares cuidadores. Los datos fueron recolectados en 2019 por entrevistas y sometidos a la codificación abierta y axial. Resultados: las familias recorrieron un largo itinerario hasta la recepción del diagnóstico del niño: detectaron alteraciones en su estado general; realizaron diversos exámenes; desconfiaron del diagnóstico por tener ya otros con el mismo en la familia; recibieron informaciones de los profesionales de la salud acerca de los cuidados necesarios y de los servicios especializados para su tratamiento; presentaron miedo, pánico y negación; y valoraron la fe en Dios en la esperanza de la mejora del niño. Consideraciones finales: el enfermero necesita desarrollar un proceso educativo con la familia para que ella se sienta preparada y capaz de cuidar a esos niños.

ABSTRACT Objective: to know the itinerary taken by the family to obtain the diagnosis of the children with special health needs. Method: a qualitative study was carried out in a pediatric inpatient unit in southern Brazil. Grounded Theory was used as a methodological reference. Sixteen (16) family caregivers participated. Data were collected in 2019 through interviews and submitted to open and axial coding. Results: the families traveled a long route until receiving the children's diagnosis: they detected changes in their general condition; performed several exams; they were suspicious of the diagnosis because other members of the family had already been diagnosed with the same conditions; received information from health professionals about the necessary care and specialized services for the treatment; showed fear, panic and denial; and they valued faith in God in the hope of improving of the children. Final considerations: nurses need to develop an educational process with the family so that they feel prepared and capable of taking care of these children.

A conjoined universal helper epitope can unveil antitumor effects of a neoantigen vaccine targeting an MHC class I-restricted neoepitope.

Personalized cancer vaccines targeting neoantigens arising from somatic missense mutations are currently being evaluated for the treatment of various cancers due to their potential to elicit a multivalent, tumor-specific immune response. Several cancers express a low number of neoantigens; in these cases, ensuring the immunotherapeutic potential of each neoantigen-derived epitope (neoepitope) is crucial. In this study, we discovered that therapeutic vaccines targeting immunodominant major histocompatibility complex (MHC) I-restricted neoepitopes require a conjoined helper epitope in order to induce a cytotoxic, neoepitope-specific CD8+ T-cell response. Furthermore, we show that the universally immunogenic helper epitope P30 can fulfill this requisite helper function. Remarkably, conjoined P30 was able to unveil immune and antitumor responses to subdominant MHC I-restricted neoepitopes that were, otherwise, poorly immunogenic. Together, these data provide key insights into effective neoantigen vaccine design and demonstrate a translatable strategy using a universal helper epitope that can improve therapeutic responses to MHC I-restricted neoepitopes.

Gating Harmonization Guidelines for Intracellular Cytokine Staining Validated in Second International Multiconsortia Proficiency Panel Conducted by Cancer Immunotherapy Consortium (CIC/CRI).

Results from the first gating proficiency panel of intracellular cytokine staining (ICS) highlighted the value of using a consensus gating approach to reduce the variability across laboratories in reported %CD8+ or %CD4+ cytokine-positive cells. Based on the data analysis from the first proficiency panel, harmonization guidelines for a consensus gating protocol were proposed. To validate the recommendations from the first panel and to examine factors that were not included in the first panel, a second ICS gating proficiency panel was organized. All participants analyzed the same set of Flow Cytometry Standard (FCS) files using their own gating protocol. An optional learning module was provided to demonstrate how to apply the previously established gating recommendations and harmonization guidelines to actual ICS data files. Eighty-three participants took part in this proficiency panel. The results from this proficiency panel confirmed the harmonization guidelines from the first panel. These recommendations addressed the (1) placement of the cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and (6) proper adjustment of the biexponential scaling. In addition, based on the results of this proficiency gating panel, two new recommendations were added to expand the harmonization guidelines: (1) inclusion of dump channel marker to gate all live and dump negative cells and (2) backgating to confirm the correct placement of gates across all populations. © 2020 International Society for Advancement of Cytometry.

Rede de apoio social da família para o cuidado da criança com paralisia cerebral / Social support network of the family for the care of children with cerebral palsy / Red de apoyo social de la familia para el cuidado del niño con parálisis cerebral

Objetivo: analisar a rede de apoio social utilizada pela família para cuidar a criança com paralisia cerebral. Método: pesquisa qualitativa, descritiva, realizada com 12 mães de crianças com paralisia cerebral atendidas em unidade de pediatria do Hospital Universitário de Rio Grande. Os dados foram coletados nos meses de agosto e setembro de 2018, por meio de entrevista semiestruturada, e submetidos à análise temática. Resultados: identificou-se a rede de apoio social formada pelos familiares, amigos, vizinhos, profissionais de saúde e ancorada pela fé em Deus. As mães foram protagonistas do cuidado e algumas não receberam nenhum tipo de apoio para os cuidados dos filhos. Conclusão: a rede de apoio social contribuiu para a superação de dificuldades da família e para o cuidado da criança, sendo fonte de apoio emocional, financeiro, ajuda com o transporte, remédios e orientação dos profissionais de saúde/enfermagem.

Objective: to know the social support network used by the family to care for children with cerebral palsy. Method: this qualitative and descriptive research was conducted with twelve mothers of children with cerebral palsy assisted at a pediatric unit of a University Hospital in Rio Grande. Data were collected in August and September 2018 through semi-structured interviews and submitted to thematic analysis. Results: we identified the social support network consisting of family, friends, neighbors, health professionals, and faith in God. Mothers were protagonists of care and some received no support for childcare. Conclusion: the social support network contributed to overcome family difficulties and childcare, being a source of emotional, financial support, help with transportation, medicine, and guidance from health/nursing professionals.

Objetivo: conocer la red de apoyo social utilizada por la familia para cuidar a niños con parálisis cerebral. Método: investigación cualitativa, descriptiva, realizada con doce madres de niños con parálisis cerebral atendidas en una unidad de pediatría de un Hospital Universitario de Río Grande. Los datos se recopilaron en agosto y septiembre de 2018 a través de entrevistas semiestructuradas y se sometieron a análisis temáticos. Resultados: identificamos que la red de apoyo social estaba formada por familiares, amigos, vecinos, profesionales de la salud y fe en Dios. Las madres fueron protagonistas de la atención y algunas no recibieron apoyo para el cuidado de los niños. Conclusión: la red de apoyo social contribuyó a superar las dificultades familiares y el cuidado de los niños, siendo una fuente de apoyo emocional y financiero, ayuda con el transporte, medicamentos y orientación de profesionales de la salud/enfermeros.

Sífilis congênita no recém-nascido: repercussões para a mãe / Congenital syphilis in newborns: repercussions for the mother / Sífilis congénita en el neonato: repercusiones para la madre

Objetivo: conhecer as repercussões do diagnóstico da Sífilis Congênita no recém-nascido para a mãe. Método: pesquisa qualitativa desenvolvida com 15 mães de recém-nascidos com Sífilis Congênita e internados em um hospital público do sul do Brasil. Os dados foram coletados por entrevistas semiestruturadas, submetidos à análise de conteúdo e aprovados pelo Comitê de Ética em Pesquisa. Resultados: as mães manifestaram sentimentos de culpa, desespero, tristeza e horror. Havia esperança de não passar a infecção para o recém-nascido. Verificou-se a reincidência da doença em mais de uma gestação. Referiram medo do estigma social e buscaram informações acerca da doença na internet, como também com médicos e enfermeiros. Conclusão: há desinformação das mães quanto à infecção da sífilis, principalmente sobre como evitar a transmissão vertical e a reinfecção. Cabe ao enfermeiro, instrumentalizar a mãe para o cuidado ao recém-nascido com Sífilis Congênita, por meio de um processo educativo que qualifique o cuidado da criança e da mãe

Objective: to learn how a diagnosis of congenital syphilis in newborns affects the mothers. Method: in this qualitative study of 15 mothers of newborns with congenital syphilis admitted to a public hospital in southern Brazil, data were collected by semistructured interview, and treated by content analysis. The study was approved by the research ethics committee. Results: the mothers expressed feelings of guilt, despair, sadness and horror. They hoped not to transmit the infection to the newborn. The disease was found to have recurred in more than one pregnancy. They reported fear of social stigma, and looked for information about the disease on the Internet, as well as from doctors and nurses. Conclusion: mothers are misinformed regarding infection by syphilis, especially on how to prevent vertical transmission and reinfection. It is up to nurses to instruct mothers on how to care for newborns with congenital syphilis, through an educational process to improve care for the child and the mother.

Objetivo: conocer las repercusiones, para la madre, del diagnóstico de sífilis congénita en el neonato. Método: investigación cualitativa desarrollada junto a 15 madres de neonatos con sífilis congénita e ingresados en un hospital público en el sur de Brasil. Se recolectaron los datos a través de entrevistas semiestructuradas y se sometieron al análisis de contenido; después fueron aprobados por el Comité de Ética en Investigación. Resultados: las madres expresaron sentimientos de culpa, desesperación, tristeza y horror. Había esperanza de no transmitir la infección al neonato. Se verificó la reincidencia de la enfermedad en más de un embarazo. Declararon el temor al estigma social y buscaron información sobre la enfermedad en internet, así como junto a médicos y enfermeros. Conclusión: las madres están desinformadas en cuanto a la infección por Sífilis, especialmente sobre cómo prevenir la transmisión vertical y la reinfección. Le toca al enfermero darle a la madre herramientas para que cuide bien al neonato con Sífilis Congénita, a través de un proceso educativo que califique el cuidado del niño y la madre

Sífilis gestacional: repercussões para a puérpera* / Sífilis gestacional: repercusiones para la puérpera / Gestational syphilis: repercussions for postpartum women

RESUMO Objetivo: conhecer as repercussões do diagnóstico de Sífilis Gestacional para a puérpera. Método: pesquisa qualitativa realizada em uma unidade de pediatria de um hospital universitário do sul do Brasil. Participaram 15 puérperas de crianças internadas no setor com diagnóstico de Sífilis Congênita. Os dados foram coletados por entrevistas semiestruturadas no primeiro semestre de 2018 e submetidos à análise de conteúdo. Resultados: obtiveram-se dados acerca do recebimento do diagnóstico, das reações frente ao diagnóstico, da influência do diagnóstico na gestação e parto e da realização do tratamento da Sífilis Gestacional. Conclusão: verificou-se desinformação das puérperas quanto à infecção da sífilis, principalmente sobre cuidados para evitar a transmissão e a reinfecção. Destaca-se o papel educativo do enfermeiro junto a essas mulheres na busca pela diminuição das (re)infecções por Sífilis.

RESUMEN: Objetivo: conocer las repercusiones del diagnóstico de Sífilis Gestacional para la puérpera. Método: investigación cualitativa realizada en una unidad de pediatría de un hospital universitario del sur de Brasil. Participaron 15 puérperas de niños internados en el sector con diagnóstico de Sífilis Congénita. Los datos se recolectaron por medio de entrevistas semiestructuradas durante el primer semestre de 2018 y se los sometió a análisis de contenido. Resultados: se obtuvieron datos acerca de cómo se recibe el diagnóstico, de las reacciones frente al mismo, de la influencia del diagnóstico en el embarazo y el parto y de la realización del diagnóstico de la Sífilis Gestacional. Conclusión: se verificó que las puérperas están desinformadas con respecto a la infección de la sífilis, principalmente sobre los cuidados para evitar su transmisión y reinfección. Se destaca el rol educativo del enfermero junto a estas mujeres en la búsqueda de reducir la cantidad de (re)infecciones por sífilis.

ABSTRACT Objective: To know the repercussions of the diagnosis of Gestational Syphilis for the postpartum woman. Method: A qualitative research carried out in a pediatric unit of a university hospital in southern Brazil. 15 postpartum women participated of children admitted to the sector diagnosed with congenital syphilis. Data was collected by semi-structured interviews in the first half of 2018 and submitted to content analysis. Results: Data was obtained about the receipt of the diagnosis, the reactions regarding the diagnosis, the influence of the diagnosis during pregnancy and childbirth and the treatment of gestational syphilis. Conclusion: Misinformation of the puerperal women regarding syphilis infection, especially regarding care to avoid transmission and reinfection. We highlight the educational role of nurses with these women in the pursuit of reducing (re)infections by syphilis.

CD27 stimulation unveils the efficacy of linked class I/II peptide vaccines in poorly immunogenic tumors by orchestrating a coordinated CD4/CD8 T cell response.

Despite their promise, tumor-specific peptide vaccines have limited efficacy. CD27 is a costimulatory molecule expressed on CD4+ and CD8+ T cells that is important in immune activation. Here we determine if a novel CD27 agonist antibody (αhCD27) can enhance the antitumor T cell response and efficacy of peptide vaccines. We evaluated the effects of αhCD27 on the immunogenicity and antitumor efficacy of whole protein, class I-restricted, and class II-restricted peptide vaccines using a transgenic mouse expressing human CD27. We found that αhCD27 preferentially enhances the CD8+ T cell response in the setting of vaccines comprised of linked class I and II ovalbumin epitopes (SIINFEKL and TEWTSSNVMEERKIKV, respectively) compared to a peptide vaccine comprised solely of SIINFEKL, resulting in the antitumor efficacy of adjuvant αhCD27 against intracranial B16.OVA tumors when combined with vaccines containing linked class I/II ovalbumin epitopes. Indeed, we demonstrate that this efficacy is both CD8- and CD4-dependent and αhCD27 activity on ovalbumin-specific CD4+ T cells is necessary for its adjuvant effect. Importantly for clinical translation, a linked universal CD4+ helper epitope (tetanus P30) was sufficient to instill the efficacy of SIINFEKL peptide combined with αhCD27, eliminating the need for a tumor-specific class II-restricted peptide. This approach unveiled the efficacy of a class I-restricted peptide vaccine derived from the tumor-associated Trp2 antigen in mice bearing intracranial B16 tumors. CD27 agonist antibodies combined with peptide vaccines containing linked tumor-specific CD8+ epitopes and tumor-specific or universal CD4+ epitopes enhance the efficacy of active cancer immunotherapy.

A simple and enzyme-free method for processing infiltrating lymphocytes from small mouse tumors for ELISpot analysis.

The ELISpot assay prevails as one of the most sensitive and meaningful assays for the detection of antigen-specific, effector immune responses. Acquisition of cellular analyte for ELISpot analysis is typically not problematic when derived from tissues enriched in lymphocytes (e.g., lymphoid organs and blood); however, cell processing becomes more difficult when lymphocytes represent only a very minor population relative to the source tissue, especially when the source tissue is in limited supply (e.g., small mouse tumors). Traditional enzymatic-based methods for dissociating tumors often result in poor yields, inconsistent lymphocyte enrichment, and can have deleterious effects on lymphocyte phenotype and function. To address these limitations, we have developed an enzyme-free protocol for processing tumor infiltrating lymphocytes (TILs) from small mouse tumors, which enables the enumeration of antigen-specific effector lymphocytes using ELISpot analysis. This procedure is predicated on the dissociation of tumor tissue using gentle agitation with a paddle blender followed by a brief in vitro culture period to remove adherent cells, as well as to revive lymphocytes from a non-responsive state. Although this method is demonstrated with mouse intracerebral tumors, we have found that this protocol is applicable to peripheral tumors and may likely extend to alternative tissue sources wherein lymphocytes exist in low numbers.

Dendritic Cells Enhance Polyfunctionality of Adoptively Transferred T Cells That Target Cytomegalovirus in Glioblastoma.

Median survival for glioblastoma (GBM) remains <15 months. Human cytomegalovirus (CMV) antigens have been identified in GBM but not normal brain, providing an unparalleled opportunity to subvert CMV antigens as tumor-specific immunotherapy targets. A recent trial in recurrent GBM patients demonstrated the potential clinical benefit of adoptive T-cell therapy (ATCT) of CMV phosphoprotein 65 (pp65)-specific T cells. However, ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8+ T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8+ T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ+, TNFα+, and CCL3+ polyfunctional, CMV-specific CD8+ T cells. These increases in polyfunctional CMV-specific CD8+ T cells correlated (R = 0.7371, P = 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256-64. ©2017 AACR.

A Supramolecular Vaccine Platform Based on α-Helical Peptide Nanofibers.

A supramolecular peptide vaccine system was designed in which epitope-bearing peptides self-assemble into elongated nanofibers composed almost entirely of alpha-helical structure. The nanofibers were readily internalized by antigen presenting cells and produced robust antibody, CD4+ T-cell, and CD8+ T-cell responses without supplemental adjuvants in mice. Epitopes studied included a cancer B-cell epitope from the epidermal growth factor receptor class III variant (EGFRvIII), the universal CD4+ T-cell epitope PADRE, and the model CD8+ T-cell epitope SIINFEKL, each of which could be incorporated into supramolecular multi-epitope nanofibers in a modular fashion.

Serum elevation of B lymphocyte stimulator does not increase regulatory B cells in glioblastoma patients undergoing immunotherapy.

Regulatory B cells that secrete IL-10 (IL-10(+) Bregs) represent a suppressive subset of the B cell compartment with prominent anti-inflammatory capacity, capable of suppressing cellular and humoral responses to cancer and vaccines. B lymphocyte stimulator (BLyS) is a key regulatory molecule in IL-10(+) Breg biology with tightly controlled serum levels. However, BLyS levels can be drastically altered upon chemotherapeutic intervention. We have previously shown that serum BLyS levels are elevated, and directly associated, with increased antigen-specific antibody titers in patients with glioblastoma (GBM) undergoing lymphodepletive temozolomide chemotherapy and vaccination. In this study, we examined corresponding IL-10(+) Breg responses within this patient population and demonstrate that the IL-10(+) Breg compartment remains constant before and after administration of the vaccine, despite elevated BLyS levels in circulation. IL-10(+) Breg frequencies were not associated with serum BLyS levels, and ex vivo stimulation with a physiologically relevant concentration of BLyS did not increase IL-10(+) Breg frequency. However, BLyS stimulation did increase the frequency of the overall B cell compartment and promoted B cell proliferation upon B cell receptor engagement. Therefore, using BLyS as an adjuvant with therapeutic peptide vaccination could promote humoral immunity with no increase in immunosuppressive IL-10(+) Bregs. These results have implications for modulating humoral responses in human peptide vaccine trials in patients with GBM.

Severe adverse immunologic reaction in a patient with glioblastoma receiving autologous dendritic cell vaccines combined with GM-CSF and dose-intensified temozolomide.

Therapeutic vaccination of patients with cancer-targeting tumor-associated antigens is a promising strategy for the specific eradication of invasive malignancies with minimal toxicity to normal tissues. However, as increasingly potent modalities for stimulating immunologic responses are developed for clinical evaluation, the risk of inflammatory and autoimmune toxicities also may be exacerbated. In this report, we describe the induction of a severe (grade 3) immunologic reaction in a patient with newly diagnosed glioblastoma (GBM) receiving autologous RNA-pulsed dendritic cell (DC) vaccines admixed with GM-CSF and administered coordinately with cycles of dose-intensified temozolomide. Shortly after the eighth administration of the admixed intradermal vaccine, the patient experienced dizziness, flushing, conjunctivitis, headache, and the outbreak of a disseminated macular/papular rash and bilateral indurated injection sites. Immunologic workup of patient reactivity revealed sensitization to the GM-CSF component of the vaccine and the production of high levels of anti-GM-CSF autoantibodies during vaccination. Removal of GM-CSF from the DC vaccine allowed continued vaccination without incident. Despite the known lymphodepletive and immunosuppressive effects of temozolomide, these observations demonstrate the capacity for the generation of severe immunologic reactivity in patients with GBM receiving DC-based therapy during adjuvant dose-intensified temozolomide.

EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss.

PURPOSE: Chimeric antigen receptor (CAR) transduced T cells represent a promising immune therapy that has been shown to successfully treat cancers in mice and humans. However, CARs targeting antigens expressed in both tumors and normal tissues have led to significant toxicity. Preclinical studies have been limited by the use of xenograft models that do not adequately recapitulate the immune system of a clinically relevant host. A constitutively activated mutant of the naturally occurring epidermal growth factor receptor (EGFRvIII) is antigenically identical in both human and mouse glioma, but is also completely absent from any normal tissues. EXPERIMENTAL DESIGN: We developed a third-generation, EGFRvIII-specific murine CAR (mCAR), and performed tests to determine its efficacy in a fully immunocompetent mouse model of malignant glioma. RESULTS: At elevated doses, infusion with EGFRvIII mCAR T cells led to cures in all mice with brain tumors. In addition, antitumor efficacy was found to be dependent on lymphodepletive host conditioning. Selective blockade with EGFRvIII soluble peptide significantly abrogated the activity of EGFRvIII mCAR T cells in vitro and in vivo, and may offer a novel strategy to enhance the safety profile for CAR-based therapy. Finally, mCAR-treated, cured mice were resistant to rechallenge with EGFRvIII(NEG) tumors, suggesting generation of host immunity against additional tumor antigens. CONCLUSION: All together, these data support that third-generation, EGFRvIII-specific mCARs are effective against gliomas in the brain and highlight the importance of syngeneic, immunocompetent models in the preclinical evaluation of tumor immunotherapies.

BLyS levels correlate with vaccine-induced antibody titers in patients with glioblastoma lymphodepleted by therapeutic temozolomide.

B lymphocyte stimulator (BLyS) is a cytokine involved in differentiation and survival of follicular B cells along with humoral response potentiation. Lymphopenia is known to precipitate dramatic elevation in serum BLyS; however, the use of this effect to enhance humoral responses following vaccination has not been evaluated. We evaluated BLyS serum levels and antigen-specific antibody titers in 8 patients undergoing therapeutic temozolomide (TMZ)-induced lymphopenia, with concomitant vaccine against a tumor-specific mutation in the epidermal growth factor receptor (EGFRvIII). Our studies demonstrate that TMZ-induced lymphopenia corresponded with spikes in serum BLyS that directly preceded the induction of anti-EGFRvIII antigen-specific antibody titers, in some cases as high as 1:2,000,000. Our data are the first clinical observation of BLyS serum elevation and greatly enhanced humoral immune responses as a consequence of chemotherapy-induced lymphopenia. These observations should be considered for the development of future vaccination strategies in the setting of malignancy.

Randomized, double-blind, Phase 1 trial of an alphavirus replicon vaccine for cytomegalovirus in CMV seronegative adult volunteers.

Development of a cytomegalovirus (CMV) vaccine is a priority. We evaluated a two component alphavirus replicon particle vaccine expressing CMV gB or a pp65/IE1 fusion protein, previously shown to induce robust antibody and cellular immune responses in mice, in a randomized, double-blind Phase 1 clinical trial in CMV seronegative subjects. Forty subjects received a lower dose (LD) or higher dose (HD) of vaccine or placebo by intramuscular or subcutaneous injection at Weeks 0, 8 and 24. The vaccine was well tolerated, with mild to moderate local reactogenicity, minimal systemic reactogenicity, and no clinically important changes in laboratory parameters. All vaccine recipients developed ex vivo, direct IFN-gamma ELISPOT responses to CMV antigens (maximal mean spot-forming cells per 10(6) PBMC in LD and HD groups of 348 and 504 for pp65, 83 and 113 for IE1, and 138 and 114 for gB), and neutralizing antibodies (maximal geometric mean titer 110 with LD and 218 with HD). Polyfunctional CD4(+) and CD8(+) T cell responses were detected by polychromatic flow cytometry. This alphavirus replicon particle vaccine was safe and induced neutralizing antibody and multifunctional T cell responses against three CMV antigens that are important targets for protective immunity.

Development and preclinical evaluation of an alphavirus replicon vaccine for influenza.

We used a propagation-defective, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the hemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/Wyoming/03/2003 (H3N2). Efficient production methods were scaled to produce pilot lots of HA VRP and NA VRP and clinical lots of HA VRP. HA VRP-induced high-titered antibody responses in mice, rabbits and rhesus macaques, as measured by ELISA or hemagglutination inhibition (HI) assays, and robust cellular immune responses in mice and rhesus macaques, as measured by IFN-gamma ELISPOT. NA VRP also induced cellular immune responses in mice. A toxicology study with HA VRP and NA VRP in rabbits showed no adverse effects in any parameter. These studies support clinical testing of alphavirus replicon vaccines for influenza.

Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1, and gB proteins.

Development of vaccines against cytomegalovirus (CMV) is an important public health priority. We used a propagation-defective, single-cycle RNA replicon vector system derived from an attenuated strain of an alphavirus, Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRP) expressing various combinations of pp65, IE1, or gB proteins of human CMV. Protein expression in VRP-infected cells was highest with single-promoter replicons expressing pp65, IE1, a pp65/IE1 fusion protein, or the extracellular domain of gB and with double-promoter replicons expressing pp65 and IE1. Protein expression was lower with double- and triple-promoter replicons expressing gB, especially the full-length form of gB. BALB/c mice immunized with VRP expressing gB developed high titers of neutralizing antibody to CMV, and mice immunized with VRP expressing pp65, IE1, or a pp65/IE1 fusion protein developed robust antigen-specific T-cell responses as measured by gamma interferon enzyme-linked immunospot assay. Three overlapping immunodominant pp65 peptides contained a nine-amino-acid sequence (LGPISGHVL) that matches the consensus binding motif for a major histocompatibility complex H2-D(d) T-cell epitope. These data provide the basis for further development and clinical evaluation of an alphavirus replicon vaccine for CMV expressing the pp65, IE1, and gB proteins.

A comparison of xylazine and medetomidine in an anesthetic combination in New Zealand White Rabbits.

A comparison was made of two anesthetic protocols for cardiothoracic surgery in rabbits. Eight male New Zealand White rabbits (2.8 to 3.2 kg) were used in a double crossover study. Each rabbit received intramuscular ketamine (35 mg/kg), xylazine (5 mg/kg), and buprenorphine (0.03 mg/kg) or ketamine (35 mg/kg), medetomidine (0.5 mg/kg), and buprenorphine (0.03 mg/kg) on alternate weeks. After intramuscular injection, each rabbit was intubated and placed on 0.75% isoflurane in 1 L O2/min. Palpebral, pedal, and righting reflexes and cardiopulmonary parameters were measured every minute for the first 10 min and every 5 min thereafter. Rabbits were monitored for 20 min of spontaneous ventilation followed by 60 min of intermittent positive pressure ventilation. Intermittent positive pressure ventilation and isoflurane then were discontinued and recovery monitored. Systolic, mean, and diastolic blood pressures were higher in the medetomidine-treated rabbits. Return of the palpebral, pedal, and righting reflexes was prolonged in the medetomidine-treated rabbits. There were no differences in heart rate, respiratory rate, return to spontaneous breathing, and time to extubation between the two groups. These results indicate medetomidine can be safely used in rabbit anesthesia, provides acceptable cardiovascular parameters, and induces a longer anesthetic period than that of xylazine.