[Review of a series of cases of Creutzfeldt-Jakob disease in a tertiary care hospital]. / Revisión de una serie de casos de enfermedad de Creutzfeldt-Jakob en un hospital de tercer nivel.

INTRODUCTION: We analysed a series of patients with sporadic Creutzfeldt-Jakob disease in our setting. AIM: The aim of this study is to describe the characteristics of our sample using the new diagnostic tools based on the most recently published criteria. MATERIAL AND METHODS: A descriptive, retrospective study was conducted using a digitalised hospital register. We identified 20 cases of the sporadic type, in the period 2012-2022: eight with a pathological diagnosis and 12 with high probability. The variables sex, age at onset, time of evolution, clinical phenotype, magnetic resonance imaging (MRI) findings, 14.3.3 protein, electroencephalogram (EEG), real-time quaking-induced prion protein conversion (RT-QuIC), autopsy, pathological phenotype and genetic diagnosis were recorded. RESULTS: Of those affected, 50% were men and 50%, women, with an age at onset of 67 years (30-83) and a mean survival time of eight months (1-11 months). Cognitive impairment was the most frequent onset symptom, followed by gait ataxia. All MRI scans with long time-lapse sequences (FLAIR and DWI) were pathological, and the pattern of diffuse cortical and basal ganglia involvement was the most frequent. Altogether, 55% of the sample had an EEG with characteristic triphasic complexes. Sixty-five per cent were positive for 14.3.3 protein in cerebrospinal fluid. Four RT QuIC studies were carried out (in 2020) and all were positive. In 40% of them a confirmatory autopsy was performed, with the MM/MV1 pattern being the most frequent. CONCLUSIONS: MRI with DWI sequences is a particularly sensitive test for the diagnosis of the disease, although its sensitivity decreases in the early stages. The high specificity and sensitivity of RT-QuIC, together with a characteristic clinical diagnosis and radiological pattern, are proposed as an alternative to the pathological definitive diagnosis.

TITLE: Revisión de una serie de casos de enfermedad de Creutzfeldt-Jakob en un hospital de tercer nivel.Introducción. Analizamos en nuestro medio una serie de pacientes con enfermedad de Creutzfeldt-Jakob esporádica. Objetivo. Describir las características de nuestra muestra haciendo uso de las nuevas herramientas diagnósticas según los últimos criterios publicados. Material y métodos. Realizamos un estudio descriptivo y retrospectivo mediante registro hospitalario digitalizado. Identificamos 20 casos del tipo esporádico, en el período 2012-2022, ocho con diagnóstico anatomopatológico y 12 con alta probabilidad. Se registraron las variables sexo, edad de inicio, tiempo de evolución, fenotipo clínico, hallazgos en la resonancia magnética (RM), proteína 14.3.3, electroencefalograma (EEG), conversión de proteína priónica inducida por agitación en tiempo real (RT-QuIC), autopsia, fenotipo anatomopatológico y diagnóstico genético. Resultados. Registramos un 50% de hombres y un 50% de mujeres afectos, con una edad de inicio de 67 años (30-83) y un tiempo de supervivencia medio de ocho meses (1-11 meses). El deterioro cognitivo fue el síntoma de inicio más frecuente, seguido de la ataxia de la marcha. Todas las RM con secuencias de tiempo de repetición largo (FLAIR y DWI) fueron patológicas, y el patrón de afectación cortical difusa y de los ganglios basales fue el más frecuente. El 55% de la muestra tuvo un EEG con complejos trifásicos característicos. El 65% mostró positiva la proteína 14.3.3 en el líquido cefalorraquídeo. Se realizaron cuatro estudios de RT-QuIC (en 2020) y todos fueron positivos. En un 40% se realizó una autopsia confirmatoria, con el patrón MM/MV1 como el más frecuente. Conclusiones. La RM con secuencias de DWI constituye una prueba especialmente sensible para el diagnóstico de la enfermedad, aunque su sensibilidad disminuye en estadios precoces. La alta especificidad y la alta sensibilidad de la RT-QuIC, junto con un diagnóstico clínico y patrón radiológico característico, se plantean como alternativa al diagnóstico de certeza anatomopatológico.

Revisión de una serie de casos de enfermedad de Creutzfeldt-Jakob en un hospital de tercer nivel / Review of a series of cases of Creutzfeldt-Jakob disease in a tertiary care hospital

Introducción Analizamos en nuestro medio una serie de pacientes con enfermedad de Creutzfeldt-Jakob esporádica. Objetivo Describir las características de nuestra muestra haciendo uso de las nuevas herramientas diagnósticas según los últimos criterios publicados. Material y métodos. Realizamos un estudio descriptivo y retrospectivo mediante registro hospitalario digitalizado. Identificamos 20 casos del tipo esporádico, en el período 2012-2022, ocho con diagnóstico anatomopatológico y 12 con alta probabilidad. Se registraron las variables sexo, edad de inicio, tiempo de evolución, fenotipo clínico, hallazgos en la resonancia magnética (RM), proteína 14.3.3, electroencefalograma (EEG), conversión de proteína priónica inducida por agitación en tiempo real (RT-QuIC), autopsia, fenotipo anatomopatológico y diagnóstico genético. Resultados Registramos un 50% de hombres y un 50% de mujeres afectos, con una edad de inicio de 67 años (30-83) y un tiempo de supervivencia medio de ocho meses (1-11 meses). El deterioro cognitivo fue el síntoma de inicio más frecuente, seguido de la ataxia de la marcha. Todas las RM con secuencias de tiempo de repetición largo (FLAIR y DWI) fueron patológicas, y el patrón de afectación cortical difusa y de los ganglios basales fue el más frecuente. El 55% de la muestra tuvo un EEG con complejos trifásicos característicos. El 65% mostró positiva la proteína 14.3.3 en el líquido cefalorraquídeo. Se realizaron cuatro estudios de RT-QuIC (en 2020) y todos fueron positivos. En un 40% se realizó una autopsia confirmatoria, con el patrón MM/MV1 como el más frecuente. Conclusiones La RM con secuencias de DWI constituye una prueba especialmente sensible para el diagnóstico de la enfermedad, aunque su sensibilidad disminuye en estadios precoces. La alta especificidad y la alta sensibilidad de la RT-QuIC, junto con un diagnóstico clínico y patrón radiológico característico, se plantean como alternativa al diagnóstico de certeza anatomopatológico. (AU)

INTRODUCTION We analysed a series of patients with sporadic Creutzfeldt-Jakob disease in our setting. AIM The aim of this study is to describe the characteristics of our sample using the new diagnostic tools based on the most recently published criteria. Material and methods. A descriptive, retrospective study was conducted using a digitalised hospital register. We identified 20 cases of the sporadic type, in the period 2012-2022: eight with a pathological diagnosis and 12 with high probability. The variables sex, age at onset, time of evolution, clinical phenotype, magnetic resonance imaging (MRI) findings, 14.3.3 protein, electroencephalogram (EEG), real-time quaking-induced prion protein conversion (RT-QuIC), autopsy, pathological phenotype and genetic diagnosis were recorded. RESULTS Of those affected, 50% were men and 50%, women, with an age at onset of 67 years (30-83) and a mean survival time of eight months (1-11 months). Cognitive impairment was the most frequent onset symptom, followed by gait ataxia. All MRI scans with long time-lapse sequences (FLAIR and DWI) were pathological, and the pattern of diffuse cortical and basal ganglia involvement was the most frequent. Altogether, 55% of the sample had an EEG with characteristic triphasic complexes. Sixty-five per cent were positive for 14.3.3 protein in cerebrospinal fluid. Four RT QuIC studies were carried out (in 2020) and all were positive. In 40% of them a confirmatory autopsy was performed, with the MM/MV1 pattern being the most frequent. CONCLUSIONS MRI with DWI sequences is a particularly sensitive test for the diagnosis of the disease, although its sensitivity decreases in the early stages. The high specificity and sensitivity of RT-QuIC, together with a characteristic clinical diagnosis and radiological pattern, are proposed as an alternative to the pathological definitive diagnosis. (AU)

[Cryptogenic new-onset super-refractory status epilepticus following SARS-CoV-2 vaccination. A case report]. / Estado epiléptico superrefractario de nueva aparición criptógeno tras vacunación contra el SARS-CoV-2. A propósito de un caso.

INTRODUCTION: New-onset super-refractory status epilepticus (NOSRSE) is a neurological emergency characterised by the development of status epilepticus in a patient without epilepsy or any known prior neurological disease and with no clear structural, toxic or metabolic cause, which recurs after 24 hours of induced coma. The most common identifiable cause is inflammatory-autoimmune. Consequently, we present a case of NOSRSE related to SARS-CoV-2 vaccination as an opportunity to investigate the dysimmune origin of this pathology. CASE REPORT: We report the case of a 40-year-old male who presented at the emergency department with fever and headache with no clear source of infection. His personal history included bacterial meningitis in childhood without any sequelae and protein S deficiency without treatment at the time, as well as vaccination with ChAdOx1 nCoV-19 21 days earlier. He was initially diagnosed with a urinary tract infection and treated with cefuroxime. Two days later, he was taken back to the emergency department with confusional symptoms and tonic-clonic seizures. He did not respond to midazolam and finally required sedation and orotracheal intubation for refractory status epilepticus. While in hospital, he required a number of lines of antiepileptic drugs, ketamine, a ketogenic diet, immunotherapy and plasmapheresis in order to successfully limit NOSRSE. The aetiological study offered normal results for serology, antineuronal antibodies in serum and cerebrospinal fluid, transthoracic echocardiography, testicular ultrasound and computed tomographic angiography. Only the control MRI scan showed a diffuse and bilateral alteration of the right hemispheric cortex and thalamic pulvinar as the only finding. CONCLUSION: It is crucial to report suspected adverse reactions associated with SARS-CoV-2 vaccination, thereby allowing continued monitoring of the risk/benefit ratio of vaccination.

TITLE: Estado epiléptico superrefractario de nueva aparición criptógeno tras vacunación contra el SARS-CoV-2. A propósito de un caso.Introducción. El estado epiléptico superrefractario de nueva aparición (NOSRSE) es una emergencia neurológica caracterizada por el desarrollo de estado epiléptico en un paciente sin epilepsia ni enfermedad neurológica previa conocida y sin clara causa estructural, tóxica o metabólica, que recurre tras 24 horas del coma inducido. La causa identificable más frecuente es la inflamatoria-autoinmune. En consecuencia, planteamos un caso de NOSRSE relacionado con la vacunación para el SARS-CoV-2 como una oportunidad de indagar el origen disinmune de esta patología. Caso clínico. Varón de 40 años que acude al servicio de urgencias refiriendo fiebre y cefalea sin claro foco infeccioso. Entre sus antecedentes personales destacamos una meningitis bacteriana en la infancia sin secuelas y un déficit de proteína S sin tratamiento en ese momento, así como vacunación con ChAdOx1 nCoV-19 21 días antes. Fue inicialmente diagnosticado de infección del tracto urinario y tratado con cefuroxima. Dos días después, se le llevó de nuevo a urgencias con cuadro confusional y crisis tonicoclónicas, sin respuesta al midazolam, y requirió finalmente sedación e intubación orotraqueal por estado epiléptico refractario. Durante su ingreso requirió múltiples líneas de antiepilépticos, quetamina, dieta cetógena, inmunoterapia y plasmaféresis para conseguir limitar el NOSRSE. El estudio etiológico ofrecía normalidad de los resultados de serología, anticuerpos antineuronales en el suero y líquido cefalorraquídeo, ecocardiografía transtorácica, ecografía testicular y angiotomografía computarizada. Únicamente la resonancia magnética de control mostró una alteración difusa y bilateral de la corteza hemisférica y pulvinar talámica derecha como único hallazgo. Conclusión. Es crucial notificar las sospechas de reacciones adversas asociadas a la vacunación frente al SARS-CoV-2, permitiendo así una supervisión continuada de la relación riesgo/beneficio de ésta.

Estado epiléptico superrefractario de nueva aparición criptógeno tras vacunación contra el SARS-CoV-2. A propósito de un caso / Cryptogenic new-onset super-refractory status epilepticus following SARS-CoV-2 vaccination. A case report

Introduction: New-onset super-refractory status epilepticus (NOSRSE) is a neurological emergency characterised by the development of status epilepticus in a patient without epilepsy or any known prior neurological disease and with no clear structural, toxic or metabolic cause, which recurs after 24 hours of induced coma. The most common identifiable cause is inflammatory-autoimmune. Consequently, we present a case of NOSRSE related to SARS-CoV-2 vaccination as an opportunity to investigate the dysimmune origin of this pathology. Case report: We report the case of a 40-year-old male who presented at the emergency department with fever and headache with no clear source of infection. His personal history included bacterial meningitis in childhood without any sequelae and protein S deficiency without treatment at the time, as well as vaccination with ChAdOx1 nCoV-19 21 days earlier. He was initially diagnosed with a urinary tract infection and treated with cefuroxime. Two days later, he was taken back to the emergency department with confusional symptoms and tonic-clonic seizures. He did not respond to midazolam and finally required sedation and orotracheal intubation for refractory status epilepticus. While in hospital, he required a number of lines of antiepileptic drugs, ketamine, a ketogenic diet, immunotherapy and plasmapheresis in order to successfully limit NOSRSE. The aetiological study offered normal results for serology, antineuronal antibodies in serum and cerebrospinal fluid, transthoracic echocardiography, testicular ultrasound and computed tomographic angiography. Only the control MRI scan showed a diffuse and bilateral alteration of the right hemispheric cortex and thalamic pulvinar as the only finding. Conclusion: It is crucial to report suspected adverse reactions associated with SARS-CoV-2 vaccination, thereby allowing continued monitoring of the risk/benefit ratio of vaccination.(AU)

Introducción: El estado epiléptico superrefractario de nueva aparición (NOSRSE) es una emergencia neurológica caracterizada por el desarrollo de estado epiléptico en un paciente sin epilepsia ni enfermedad neurológica previa conocida y sin clara causa estructural, tóxica o metabólica, que recurre tras 24 horas del coma inducido. La causa identificable más frecuente es la inflamatoria-autoinmune. En consecuencia, planteamos un caso de NOSRSE relacionado con la vacunación para el SARS-CoV-2 como una oportunidad de indagar el origen disinmune de esta patología. Caso clínico: Varón de 40 años que acude al servicio de urgencias refiriendo fiebre y cefalea sin claro foco infeccioso. Entre sus antecedentes personales destacamos una meningitis bacteriana en la infancia sin secuelas y un déficit de proteína S sin tratamiento en ese momento, así como vacunación con ChAdOx1 nCoV-19 21 días antes. Fue inicialmente diagnosticado de infección del tracto urinario y tratado con cefuroxima. Dos días después, se le llevó de nuevo a urgencias con cuadro confusional y crisis tonicoclónicas, sin respuesta al midazolam, y requirió finalmente sedación e intubación orotraqueal por estado epiléptico refractario. Durante su ingreso requirió múltiples líneas de antiepilépticos, quetamina, dieta cetógena, inmunoterapia y plasmaféresis para conseguir limitar el NOSRSE. El estudio etiológico ofrecía normalidad de los resultados de serología, anticuerpos antineuronales en el suero y líquido cefalorraquídeo, ecocardiografía transtorácica, ecografía testicular y angiotomografía computarizada. Únicamente la resonancia magnética de control mostró una alteración difusa y bilateral de la corteza hemisférica y pulvinar talámica derecha como único hallazgo. Conclusión: Es crucial notificar las sospechas de reacciones adversas asociadas a la vacunación frente al SARS-CoV-2, permitiendo así una supervisión continuada de la relación riesgo/beneficio de ésta.(AU)

Antigen cross-presentation and T-cell cross-priming in cancer immunology and immunotherapy.

Dendritic cells (DCs) are the main professional antigen-presenting cells for induction of T-cell adaptive responses. Cancer cells express tumor antigens, including neoantigens generated by nonsynonymous mutations, but are poor for antigen presentation and for providing costimulatory signals for T-cell priming. Mounting evidence suggests that antigen transfer to DCs and their surrogate presentation on major histocompatibility complex class I and II molecules together with costimulatory signals is paramount for induction of viral and cancer immunity. Of the great diversity of DCs, BATF3/IRF8-dependent conventional DCs type 1 (cDC1) excel at cross-presentation of tumor cell-associated antigens. Location of cDC1s in the tumor correlates with improved infiltration by CD8+ T cells and tumor-specific T-cell immunity. Indeed, cDC1s are crucial for antitumor efficacy using checkpoint inhibitors and anti-CD137 agonist monoclonal antibodies in mouse models. Enhancement and exploitation of T-cell cross-priming by cDC1s offer opportunities for improved cancer immunotherapy, including in vivo targeting of tumor antigens to internalizing receptors on cDC1s and strategies to increase their numbers, activation and priming capacity within tumors and tumor-draining lymph nodes.

Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates.

Coloboma de cristalino / Crystalline coloboma

CASO CLÍNICO: Paciente mujer de 73 años diagnosticada de coloboma de cristalino tras ser derivada a consulta para cirugía de catarata. DISCUSIÓN: La incidencia de coloboma en España por cada 10.000 nacimientos es de 0,5, en Francia de 1,4, en Estados Unidos de 2,6 y en China de 7,5. El diagnóstico es clínico mediante visualización directa. El pronóstico visual depende de la extensión y gravedad del defecto. El tratamiento de la catarata se realizó mediante la técnica convencional de facoemulsificación más lente intraocular de cápsula posterior (FACO + LIO CP) con ayuda de anillo de tensión capsular

CASE REPORT: We present a seventy-three year-old female diagnosed with lens coloboma when she was going to be operated on for cataracts. DISCUSSION: The incidence of congenital coloboma of the eye is estimated to be 0.5 per 10000 newborns in Spain, 1.4 in France, 2.6 in United States, and 7.5 in China. The diagnosis is clinical by observing the defect. The visual prognosis depends on the extent and severity of the coloboma. The treatment of the cataract was by phacoemulsification with an intraocular lens using a capsular tension ring

Crystalline coloboma.

CASE REPORT: We present a seventy-three year-old female diagnosed with lens coloboma when she was going to be operated on for cataracts. DISCUSSION: The incidence of congenital coloboma of the eye is estimated to be 0.5 per 10000 newborns in Spain, 1.4 in France, 2.6 in United States, and 7.5 in China. The diagnosis is clinical by observing the defect. The visual prognosis depends on the extent and severity of the coloboma. The treatment of the cataract was by phacoemulsification with an intraocular lens using a capsular tension ring.

Thyrocytes from autoimmune thyroid disorders produce the chemokines IP-10 and Mig and attract CXCR3+ lymphocytes.

To better understand the selective migration of lymphocytes in autoimmune thyroid disorders (AITDs), we analyzed thyroid samples and demonstrated an enhanced expression of the chemokines interferon (IFN)-inducible protein (Ip)-10 and regulated on activation normal T lymphocyte expressed and secreted (RANTES) in thyroids from AITD patients. Ip-10 and monokine induced by IFN-gamma (Mig) were expressed in vivo in thyroid follicular cells (TFCs) from AITD thyroids. Interestingly, Ip-10 mRNA, although not basally detected in cultured TFCs, was strongly induced by IFN-gamma and synergistically increased by TNF-alpha addition. Furthermore, high levels of Ip-10 protein were detected in the supernatants of IFN-gamma-stimulated TFCs. Likewise, Mig protein was strongly induced in TFCs by the same stimuli as Ip-10. Unlike Ip-10 and Mig, the expression of RANTES was induced mainly by TNF-alpha. In addition, intrathyroidal lymphocytes from AITD patients showed higher expression of CXCR3, CCR2, and CCR5 chemokine receptors than autologous peripheral blood lymphocytes. T lymphoblasts expressing CXCR3 showed an increased migration to supernatants from stimulated TFCs, which was abolished by specific antibodies to the chemokines Ip-10 and Mig, as well as to their receptor CXCR3. Taken together, these data suggest a potential role of TFCs, through the production of the chemokines Ip-10, Mig and RANTES, in regulating the recruitment of specific subsets of activated lymphocytes in AITDs.

Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors.

CD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-alpha synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-alpha was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via FcepsilonRI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.

Cyclosporin A inhibits CD69 expression induced on synovial fluid and peripheral blood lymphocytes by interleukin 15.

OBJECTIVE: To study the modulation of CD69 expression on peripheral blood (PB) and synovial fluid (SF) lymphocytes by interleukin 15 (IL-15) and several other cytokines and chemokines widely detected in the rheumatoid microenvironment. The effect of cyclosporin A (CSA) or methotrexate (MTX) in the cytokine mediated regulation of CD69 was analyzed. METHODS: CD69 expression on lymphocytes was assessed by flow cytometry after incubation with different cytokines, chemokines, phorbol myristate acetate, or calcium ionophore in the presence or absence of CSA, MTX, or both. The effect of IL-15 and SF supernatants in maintaining CD69 expression on SF lymphocytes was also assessed. IL-15 levels in SF supernatants were measured by ELISA. RESULTS: IL-15 induced the greatest upregulation of CD69 expression on PB lymphocytes in a time and dose dependent manner. IL-15 was able to maintain a high CD69 expression on SF lymphocytes. SF supernatants from rheumatoid arthritis (RA), which contain significant amounts of IL-15, also reversed the CD69 downregulation of SF lymphocytes in culture. CSA, but not MTX, inhibited the CD69 upregulation mediated by IL-15 both in PB and SF lymphocytes. CONCLUSION: IL-15 appears to be responsible, at least in part, for the high CD69 expression on lymphocytes from the rheumatoid microenvironment. Consistent with the virtual absence of lymphocyte derived cytokines in RA synovium, the prevention of IL-15 mediated CD69 upregulation on lymphocytes may explain the effect of CSA in the treatment of RA.

The tyrosine kinase PYK-2/RAFTK regulates natural killer (NK) cell cytotoxic response, and is translocated and activated upon specific target cell recognition and killing.

The compartmentalization of plasma membrane proteins has a key role in regulation of lymphocyte activation and development of immunity. We found that the proline-rich tyrosine kinase-2 (PYK-2/RAFTK) colocalized with the microtubule-organizing center (MTOC) at the trailing edge of migrating natural killer (NK) cells. When polyclonal NK cells bound to K562 targets, PYK-2 translocated to the area of NK-target cell interaction. The specificity of this process was assessed with NK cell clones bearing activatory or inhibitory forms of CD94/NKG2. The translocation of PYK-2, MTOC, and paxillin to the area of NK-target cell contact was regulated upon specific recognition of target cells through NK cell receptors, controlling target cell killing. Furthermore, parallel in vitro kinase assays showed that PYK-2 was activated in response to signals that specifically triggered its translocation and NK cell mediated cytotoxicity. The overexpression of both the wt and a dominant-negative mutant of PYK-2, but not ZAP-70 wt, prevented the specific translocation of the MTOC and paxillin, and blocked the cytotoxic response of NK cells. Our data indicate that subcellular compartmentalization of PYK-2 correlates with effective signal transduction. Furthermore, they also suggest an important role for PYK-2 on the assembly of the signaling complexes that regulate the cytotoxic response.

Phenotypic and functional characteristics of hematopoietic cell lineages in CD69-deficient mice.

AIM/CD69 is the earliest leukocyte activation antigen and is expressed mainly by activated T, B, and natural killer (NK) cells. It is also constitutively expressed by platelets, by bone marrow myeloid precursors, and by small subsets of resident lymphocytes in the secondary lymphoid tissues. The engagement of CD69 by specific antibodies induces intracellular signals, including Ca(++) flux, cytokine synthesis, and cell proliferation. To investigate the physiological relevance of CD69, we generated mice deficient in CD69 (CD69-/-) by gene targeting in embryonic stem cells. CD69 (-/-) mice showed largely normal hematopoietic cell development and normal T-cell subpopulations in thymus and periphery. Furthermore, studies of negative- and positive-thymocyte selection using a T-cell receptor transgenic model demonstrated that these processes were not altered in CD69 (-/-) mice. In addition, natural killer and cytotoxic T lymphocyte cells from CD69-deficient mice displayed cytotoxic activity similar to that of wild-type mice. Interestingly, B-cell development was affected in the absence of CD69. The B220(hi)IgM(neg) bone marrow pre-B cell compartment was augmented in CD69 (-/-) mice. In addition, the absence of CD69 led to a slight increase in immunoglobulin (Ig) G2a and IgM responses to immunization with T-dependent and T-independent antigens. Nevertheless, CD69-deficient lymphocytes had a normal proliferative response to different T-cell and B-cell stimuli. Together, these observations indicate that CD69 plays a role in B-cell development and suggest that the putative stimulatory activity of this molecule on bone marrow-derived cells may be replaced in vivo by other signal transducing receptors.

Signaling through CD43 induces natural killer cell activation, chemokine release, and PYK-2 activation.

Natural killer (NK) cell activation is the result of a balance between positive and negative signals triggered by specific membrane receptors. We report here the activation of NK cells induced through the transmembrane glycoprotein CD43 (leukosialin, sialophorin). Engagement of CD43 by specific antibodies stimulated the secretion of the chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, which was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, signaling through CD43 increased the cytotoxic activity of NK cells and stimulated an increase in the tyrosine kinase activity in antiphosphotyrosine immune complexes of NK cell lysates. PYK-2 was identified among the tyrosine kinase proteins that become activated. Hence, PYK-2 activation was observed after 20 minutes of CD43 stimulation, reached a maximum after 45 to 60 minutes, and decreased to almost basal levels after 120 minutes of treatment. Together, these results demonstrate the role of CD43 as an activation molecule able to transduce positive activation signals in NK cells, including the regulation of chemokine synthesis, killing activity, and tyrosine kinase activation.

Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.

Activation of peripheral blood T cells by interaction and migration through endothelium: role of lymphocyte function antigen-1/intercellular adhesion molecule-1 and interleukin-15.

Cell adhesion molecules have a key role in the migration of T cells to inflammatory foci. However, the effect of the endothelial-lymphocyte interaction on the activation of the latter cells remains unresolved. We have studied the effect of resting and stimulated endothelial cells (ECs) on the activation of peripheral blood T cells (PBTLs), as assessed by the expression of CD69 and CD25 activation antigens. The incubation of PBTLs with tumor necrosis factor-alpha-activated EC monolayers, either alive or fixed, induced the expression of CD69 but not CD25, preferentially in the CD8(+) CD45RO+ cell subset. Furthermore, it induced the production of cytokines such as IFN-gamma, but not that of interleukin-2 (IL-2) and IL-4. EC treated with other stimuli such as IL-1beta, IFN-gamma, or lipopolysaccharide also showed the same proactivatory effect on T cells. Lymphocyte activation was almost completely inhibited by blocking anti-CD18 and anti-intercellular adhesion molecule-1 (anti-ICAM-1) monoclonal antibodies (MoAbs), but only slightly affected by MoAbs against CD49d, vascular cell adhesion molecule-1, and anti-IL-15. In addition, the interaction of PBTL with immobilized ICAM-1 induced CD69 expression in the same memory T-cell subset. IL-15 induced T-cell activation with expression of CD69 and CD25, and production of IFN-gamma, and its effect was additive with that triggered by cell adhesion to either EC or immobilized ICAM-1. The transmigration of PBTLs through either confluent EC monolayers or ICAM-1-coated membranes also induced efficiently the expression of CD69. When IL-15 was used as chemoattractant in these assays, a further enhancement in CD69 expression was observed in migrated cells. Together these results indicate that stimulated endothelium may have an important role in T-cell activation, through the lymphocyte function antigen-1/ICAM-1 pathway, and that IL-15 efficiently cooperates in this phenomenon. These observations could account for the abundance of CD69(+) cells in the lymphocytic infiltrates of several chronic inflammatory diseases.

The two poles of the lymphocyte: specialized cell compartments for migration and recruitment.

Chemotaxis, the directed migration of leukocytes towards a chemoattractant gradient, is a key phenomenon in the immune response. During lymphocyte-endothelial and -extracellular matrix interactions, chemokines induce the polarization of T lymphocytes, with generation of specialized cell compartments. The chemokine receptors involved in detection of the chemoattractant gradients concentrate at the leading edge (advancing front or anterior pole) of the cell. The adhesion molecules ICAM-1, -3, CD44 and CD43 redistribute to the uropod, an appendage at the posterior pole of migrating T lymphocyte that protrudes from the contact area with endothelial or extracellular matrix substrates. Whereas chemokine receptors sense the direction of migration, the uropod is involved in the recruitment of bystander leukocytes through LFA-1/ICAM-dependent cell-cell interactions. While beta-actin concentrates preferentially at the cell's leading edge, the motor protein myosin II and a microtubule organizing center (MTOC) are packed in the uropod. The actin-binding protein moesin, which belongs to the ERM family of ezrin, radixin and moesin, redistributes to the distal portion of uropods and physically interacts with ICAM-3, CD44 and CD43, thus acting as a physical link between the membrane molecules and the actin cytoskeleton. Moreover, the moesin-ICAM-3 association correlates with the degree of cell polarity. The redistribution of the chemokine receptors and adhesion molecules to opposite poles of the cell in response to a chemoattractant gradient may guide cell migration and cell-cell interactions during lymphoid cell trafficking in immune and inflammatory responses.

Determination of copper and arsenic in refined beet sugar by stripping voltammetry without sample pretreatment.

Copper and arsenic have been analysed in refined beet sugar at the microgram kg-1 level by anodic stripping voltammetry (copper) and cathodic stripping voltammetry (arsenic) in the differential-pulse mode (DPASV and DPCSV) at a hanging mercury drop electrode (HMDE). DPCSV measurements of arsenic are based on its accumulation onto the HMDE as an intermetallic Cu-As compound followed by the reduction of As0 to arsine in hydrochloric acid medium. Measurements were directly carried out on untreated sugar solutions. The performance of the procedures was compared with electrothermal atomic absorption spectrometry and stripping voltammetry applied to digested sugar samples, and showed in general better accuracy. The procedures were applied to the determination of these toxic elements in commercial beet sugar samples of concentrations below 50 micrograms kg-1 copper and 15 micrograms kg-1 arsenic were found, which are much lower that those allowed by the current regulations.

Polarization of chemokine receptors to the leading edge during lymphocyte chemotaxis.

Leukocyte migration in response to cell attractant gradients or chemotaxis is a key phenomenon both in cell movement and in the inflammatory response. Chemokines are quite likely to be the key molecules directing migration of leukocytes that involve cell polarization with generation of specialized cell compartments. The precise mechanism of leukocyte chemoattraction is not known, however. In this study, we demonstrate that the CC chemokine receptors CCR2 and CCR5, but not cytokine receptors such as interleukin (IL)-2Ralpha, IL-2Rbeta, tumor necrosis factor receptor 1, or transforming growth factor betaR, are redistributed to a pole in T cells that are migrating in response to chemokines. Immunofluorescence and confocal microscopy studies show that the chemokine receptors concentrate at the leading edge of the cell on the flattened cell-substratum contact area, induced specifically by the signals that trigger cell polarization. The redistribution of chemokine receptors is blocked by pertussis toxin and is dependent on cell adhesion through integrin receptors, which mediate cell migration. Chemokine receptor expression on the leading edge of migrating polarized lymphocytes appears to act as a sensor mechanism for the directed migration of leukocytes through a chemoattractant gradient.