RESUMO
We present the design simulation and characterization of a quantum cascade detector operating at 4.3µm wavelength. Array integration and packaging processes were investigated. The device operates in the 4.3µm CO2 absorption region and consists of 64 pixels. The detector is designed fully compatible to standard processing and material growth methods for scalability to large pixel counts. The detector design is optimized for a high device resistance at elevated temperatures. A QCD simulation model was enhanced for resistance and responsivity optimization. The substrate illuminated pixels utilize a two dimensional Au diffraction grating to couple the light to the active region. A single pixel responsivity of 16mA/W at room temperature with a specific detectivity D* of 5â 107 cmHz/W was measured.
RESUMO
Strongly decreased leucocyte counts and a reduced CD4/CD8 T cell ratio in the cerebrospinal fluid (CSF) of natalizumab (NZB)-treated multiple sclerosis (MS) patients may have implications on central nervous (CNS) immune surveillance. With regard to NZB-associated progressive multi-focal leucoencephalopathy, we aimed at delineating a relationship between free NZB, cell-bound NZB, adhesion molecule (AM) expression and the treatment-associated shift in the CSF T cell ratio. Peripheral blood (PB) and CSF T cells from 15 NZB-treated MS patients, and CSF T cells from 10 patients with non-inflammatory neurological diseases and five newly diagnosed MS patients were studied. Intercellular adhesion molecule-1 (ICAM-1), leucocyte function antigen-1 (LFA-1), very late activation antigen-4 (VLA-4), NZB saturation levels, and T cell ratios were analysed by flow cytometry. NZB concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Lower NZB saturation levels (P<0.02) and a higher surface expression of ICAM-1 and LFA-1 (P<0.001) were observed on CSF CD8 T cells. CSF T cell ratios (0.3-2.1) and NZB concentrations (0.01-0.42 µg/ml) showed a pronounced interindividual variance. A correlation between free NZB, cell-bound NZB or AM expression levels and the CSF T cell ratio was not found. Extremely low NZB concentrations and a normalized CSF T cell ratio were observed in one case. The differential NZB saturation and AM expression of CSF CD8 T cells may contribute to their relative enrichment in the CSF. The reduced CSF T cell ratio appeared sensitive to steady-state NZB levels, as normalization occurred quickly. The latter may be important concerning a fast reconstitution of CNS immune surveillance.
Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Monoclonais Humanizados/uso terapêutico , Relação CD4-CD8 , Líquido Cefalorraquidiano/citologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Adulto , Moléculas de Adesão Celular/metabolismo , Monitoramento de Medicamentos , Feminino , Humanos , Imunofenotipagem , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Natalizumab , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
BACKGROUND: More and more patients with multiple sclerosis (MS) switch from natalizumab to fingolimod because of the risk of progressive multifocal leukoencephalopathy. The duration of the treatment holiday is still under debate referring to a possible recurrence of disease activity. AIM OF THE STUDY: The aim of this study was to evaluate the prognostic value of natalizumab saturation on T cells for the recurrence of clinical and radiological disease activity. METHODS: Cell surface-bound natalizumab saturation (in%) of CD8+ and CD4+ T cells from five patients with MS was determined before initiation of fingolimod by flow cytometry and related to clinical and MRI outcome during a 6-month follow-up. RESULTS: In two patients with either clinical or radiological disease activity, the natalizumab saturation on CD8+ and CD4+ T cells was <30%. In contrast, the remaining three patients with absence of disease activity had a median natalizumab saturation of 70% (range 59-79%) on CD4+ and 66% (range 52-68%) on CD8+ T cells. CONCLUSIONS: The data of this pilot study indicate that clinical and radiological disease activity is closely linked to natalizumab saturation at the time point of switch. The determination of natalizumab saturation may be an essential tool to monitor cessation of natalizumab treatment.
Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/metabolismo , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Adulto , Feminino , Cloridrato de Fingolimode , Humanos , Imunossupressores/uso terapêutico , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/patologia , Natalizumab , Propilenoglicóis/uso terapêutico , Esfingosina/análogos & derivados , Esfingosina/uso terapêutico , Fatores de TempoRESUMO
An altered expression pattern of adhesion molecules (AM) on the surface of immune cells is a premise for their extravasation into the central nervous system (CNS) and the formation of acute brain lesions in multiple sclerosis (MS). We evaluated the impact of glatiramer acetate (GA) on cell-bound and soluble AM in the peripheral blood of patients with relapsing-remitting MS (RRMS). Fifteen patients treated de novo with GA were studied on four occasions over a period of 12 months. Surface levels of intracellular cell adhesion molecule (ICAM)-1, ICAM-3, lymphocyte function-associated antigen (LFA)-1 and very late activation antigen (VLA)-4 were assessed in T cells (CD3(+) CD8(+) , CD3(+) CD4(+) ), B cells, natural killer (NK) cells, natural killer T cells (NK T) and monocytes by five-colour flow cytometry. Soluble E-selectin, ICAM-1, ICAM-3, platelet endothelial cell adhesion molecule (PECAM)-1, P-selectin and vascular cell adhesion molecule (VCAM)-1 were determined with a fluorescent bead-based immunoassay. The pro-migratory pattern in RRMS was verified by comparison with healthy controls and was characterized by up-regulation of LFA-1 (CD3(+) CD4(+) T cells, B cells), VLA-4 (CD3(+) CD8(+) T cells, NK cells), ICAM-1 (B cells) and ICAM-3 (NK cells). Effects of GA treatment were most pronounced after 6 months and included attenuated levels of LFA-1 (CD3(+) CD4(+) ) and VLA-4 (CD3(+) CD4(+) , CD3(+) CD8(+) , NK, NK T, monocytes). Further effects included lowering of ICAM-1 and ICAM-3 levels in almost all immune cell subsets. Soluble AM levels in RRMS did not differ from healthy controls and remained unaltered after GA treatment. The deregulated pro-migratory expression profile of cell-bound AM is altered by GA treatment. While this alteration may contribute to the beneficial action of the drug, the protracted development and unselective changes indicate more secondary immune regulatory phenomena related to these effects.
Assuntos
Moléculas de Adesão Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Imunossupressores/farmacologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Peptídeos/farmacologia , Adulto , Estudos de Casos e Controles , Moléculas de Adesão Celular/sangue , Membrana Celular/metabolismo , Feminino , Acetato de Glatiramer , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/metabolismoRESUMO
Recently, the disappearance of oligoclonal bands (OCBs) from the cerebrospinal fluid (CSF) of a few natalizumab-treated patients with multiple sclerosis (MS) has been reported. This is interesting since CSF-restricted OCB are believed to persist in MS. We pooled CSF data from 14 MS centers to obtain an adequate sample size for investigating the suspected changes in central nervous system (CNS)-restricted humoral immune activities in the context of natalizumab therapy. In a retrospective chart analysis, CSF parameters of blood-CSF barrier integrity and intrathecal IgG production from 73 natalizumab-treated MS patients requiring a diagnostic puncture for exclusion of progressive multifocal leukoencephalopathy were compared with CSF data obtained earlier in the course of disease before natalizumab therapy. At the time of repeat lumbar puncture, local IgG production (according to Reibergram) was significantly reduced (p < 0.0001) and OCB had disappeared in 16% of the patients. We therefore conclude that natalizumab therapy interferes with intrathecal antibody production at least in a significant number of patients.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Formação de Anticorpos/efeitos dos fármacos , Linfócitos B/imunologia , Imunoglobulina G/líquido cefalorraquidiano , Esclerose Múltipla/líquido cefalorraquidiano , Bandas Oligoclonais/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Linfócitos B/efeitos dos fármacos , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Natalizumab , Bandas Oligoclonais/efeitos dos fármacos , Bandas Oligoclonais/imunologia , Estudos Retrospectivos , Adulto JovemRESUMO
Natalizumab is a humanized monoclonal antibody directed against the alpha-4 integrin subunit of very late activation antigen-4 (VLA-4). Natalizumab neutralizing antibodies (NAB) have been found to significantly reduce beneficial effects of natalizumab treatment in multiple sclerosis. We investigated interactions of NAB with natalizumab by serial measurements of alpha-4 integrin levels on peripheral blood mononuclear cells using flow cytometry. In addition, serum concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1), the endothelial ligand of VLA-4, and serum NAB were serially determined. Natalizumab infusion led to a transient reduction in alpha-4 integrin levels on immune cells and serum sVCAM-1 levels along with serum negativity of NAB lasting for a few days post-infusion. Apparently, the high-dose effect of freshly infused natalizumab resulted in a transient neutralization of NAB possibly involving a transient therapeutic effectiveness.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/imunologia , Adulto , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/imunologia , Feminino , Citometria de Fluxo , Humanos , Integrina alfa4beta1/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Natalizumab , Molécula 1 de Adesão de Célula Vascular/sangueRESUMO
BACKGROUND: The diagnosis of the isolated leptomeningeal involvement of a primary central nervous system B-cell lymphoma without parenchyma lesions may be difficult. Patients with leptomeningeal meningeosis lymphomatosa can present with various neurologic deficits. AIMS OF THE STUDY: To demonstrate the impact of cerebrospinal fluid (CSF) flow cytometry in the diagnosis of an isolated leptomeningeal manifestation of B-cell lymphoma by presenting an interesting case report. METHODS: Flow cytometric analysis of B-cell monoclonality of the CSF was performed as complementary diagnostic procedure in addition to CSF cytology. Final diagnosis was confirmed by necropsy. RESULTS: We suspected isolated leptomeningeal manifestation of B-cell lymphoma with palsy of the VI and VII cranial nerves in a 79-year-old male, because of mononuclear pleocytosis in CSF. Interestingly, the decisive diagnostic hint was given by implementation of flow cytometry of the CSF. Diagnosis was confirmed by postmortem autopsy. CONCLUSION: Our case shows that flow cytometry of the CSF in addition to conventional CSF cytology has the potential to accelerate diagnosis of lymphomeningeal infiltration of B-cell lymphoma.
Assuntos
Neoplasias do Sistema Nervoso Central/patologia , Citometria de Fluxo , Linfoma não Hodgkin/patologia , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/secundário , Idoso , Neoplasias do Sistema Nervoso Central/líquido cefalorraquidiano , Técnicas Citológicas , Humanos , Linfoma não Hodgkin/líquido cefalorraquidiano , Masculino , Neoplasias Meníngeas/líquido cefalorraquidiano , Necrose/diagnósticoRESUMO
BACKGROUND: Natalizumab is the first monoclonal antibody therapy approved for multiple sclerosis (MS). Its therapeutic mechanism is the blockade of the α4-integrin subunit of the adhesion molecule (AM) very late activation antigen-4 (VLA-4), which leads to an inhibition of immune cell extravasation into the central nervous system (CNS). METHODS: We investigated changes in the expression levels of unblocked α4-integrin and further AM (intercellular adhesion molecule-1, -2, -3 (cICAM-1, -2, -3), leukocyte function associated antigen-1 (LFA-1)) on peripheral blood mononuclear cells (PBMC) determined by flow cytometry from 25 patients with MS before the first natalizumab infusion and before the fourth infusion. In 15 MS patients AM expression was evaluated every 3 months over 1 year. RESULTS: We found a significant decrease (p < 0.0001) of unblocked α4-integrin cell surface expression on all investigated PBMC subsets (T cells -61.7%, B cells -69.1%, monocytes/macrophages -46.4%) in the blood of MS patients after 3 months of natalizumab treatment. Moreover, a continuous decrease (p < 0.05) of unblocked α4-integrin expression levels was seen after 3, 6, 9, and 12 months. As a secondary effect, expression levels of the other investigated AM were differentially affected. CONCLUSIONS: Results show a sustained decrease of unblocked α4-integrin expression not only in all patients but also in all investigated PBMC subsets. This probably results in a continuously decreasing transmigration of PBMC into the CNS and may explain the improved clinical efficacy in the second treatment year and also the increasing risk of progressive multifocal leukoencephalopathy during long-term natalizumab therapy. We conclude that AM expression profiles are promising candidates for the development of a biomarker system to determine both natalizumab treatment response and patients at risk for opportunistic CNS infections.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Moléculas de Adesão Celular/sangue , Fatores Imunológicos/administração & dosagem , Leucócitos Mononucleares/efeitos dos fármacos , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Adolescente , Adulto , Anticorpos Monoclonais Humanizados , Antígenos CD/sangue , Áustria , Biomarcadores/sangue , Criança , Feminino , Citometria de Fluxo , Humanos , Integrina alfa4/sangue , Molécula 1 de Adesão Intercelular/sangue , Leucócitos Mononucleares/imunologia , Antígeno-1 Associado à Função Linfocitária/sangue , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/imunologia , Natalizumab , Estudos Prospectivos , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Recent data support a key role of B cells in the pathogenesis of multiple sclerosis. Due to the pronounced effect of cladribine on memory B cells, we initiated an immune phenotyping study, which included monitoring of memory B cells of patients newly assigned to this treatment option. A patient with ongoing disease activity in the first year of cladribine after a long-standing fingolimod treatment caught our attention. OBJECTIVE: To report about differences in the immune phenotype of the case compared to patients without disease activity and to discuss possible causes for the deviations as caveats regarding treatment sequelae. METHODS: Clinical data and immune phenotyping data collected at baseline (before treatment) and after three, six and ten/twelve months after cladribine initiation were compared between our case and six patients with a stable disease course (controls). RESULTS: Both, the case and controls showed similar reductions of memory B cells in response to cladribine. The case however, showed an accelerated repopulation dynamic of naïve B cells with an almost 3-fold hyperrepopulation compared to baseline levels, and lower pre-treatment levels of CD4+ and CD8+ T cells and memory B cells compared to controls. CONCLUSION: We propose a prolonged pre-treatment with fingolimod as possible cause for the lack of response to cladribine. Autoreactive cells sequestrated within lymph nodes may have evaded cladribine depletion on top of a delay of recirculating regulatory T cells. In addition, we want to raise awareness of the importance of monitoring T and B cells for bridging the current lack of evidence regarding sequencing therapies in the real life setting.
Assuntos
Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Cladribina/uso terapêutico , Cloridrato de Fingolimode/uso terapêutico , Humanos , Imunossupressores/efeitos adversos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológicoRESUMO
Recent evidence exists that the expression of the Leu-3/T4 antigen is not restricted to thymus-derived lymphocytes but can also be detected on mononuclear phagocytes and epidermal Langerhans cells (LC). When searching for the presence of Leu-3/T4 antigen-bearing cells in tissue sections of a variety of inflammatory and neoplastic skin disorders, we observed quantitative and qualitative differences in the intensity of anti-Leu-3a labeling of epidermal dendritic cells. Reasoning that Leu-3/T4 expression by these cells might be a dynamic event, we compared the anti-Leu-3a LC staining pattern in clinically normal-appearing skin (CNAS) with the expression of this antigen on epidermal dendritic cells in a variety of skin disorders. For this purpose, 4-microns cryostat sections were exposed to the monoclonal anti-Leu-3a reagent and antibody binding was visualized by a sensitive 4-step immunoperoxidase technique. Within CNAS, Leu-3a+ dendritic epidermal cells were visualized at the threshold of detectability. Immunoelectron microscopic studies confirmed the LC nature of these cells. In sharp contrast to CNAS, strong and prominent anti-Leu-3a LC labeling was almost invariably encountered in biopsy specimens from patients with cutaneous T-cell lymphoma and various inflammatory conditions but not in proliferative disorders of resident skin cells. Whereas in CNAS the density of T6+ epidermal dendritic cells greatly exceeded that of anti-Leu-3a-reactive dendritic cells, these differences were less pronounced in diseased skin. Our results: confirm earlier observations that epidermal LC may bear Leu-3/T4 antigens; and in addition, suggest that the degree of Leu-3/T4 expression is regulated by signals from inflammatory cells. The induction of class II alloantigen receptors on class II alloantigen-bearing LC may represent an important regulation mechanism of antigen-presenting cell function.
Assuntos
Antígenos de Superfície/análise , Células de Langerhans/imunologia , Dermatopatias/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Antígenos de Diferenciação de Linfócitos T , Antígenos de Superfície/imunologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Neutralizing antibodies (NAb) against interferon-beta (IFNß) affect its treatment efficacy. So far, there are no anti-NAb strategies available. OBJECTIVES: To investigate if the repeated administration of high-dose IFNß-1b intravenous in NAb positive multiple sclerosis (MS) patients induces tolerance and establishes IFNß bioavailability as measured by the induction of myxovirus protein A (MxA). METHODS: Nine MS patients with NAb titers >500 10-fold reduction units (TRU) received 1500µg IFNß-1b intravenously once weekly over three months. Blood samples were collected at screening, monthly during the treatment period (before and four hours after IFNß administration), and at follow-up after 6 months for determination of NAbs and MxA expression. RESULTS: Median NAb titer at baseline was 1429TRU. NAb titers determined before each infusion did not significantly change over the treatment period and were not different at follow-up compared to baseline. However, NAb titers were significantly decreased four hours after IFNß infusions (by roughly 50%) and MxA mRNA levels were significantly elevated reaching a median value of 206. CONCLUSIONS: Weekly intravenous administration of IFNß in patients with high NAb titers established its bioavailability, but failed to induce tolerance towards IFNß.
RESUMO
Inflammatory serum parameters are intensely investigated in the search of biomarkers for disease activity and treatment response in multiple sclerosis (MS). A reason for contradictory results might be the timing of blood collection for analyzing serum concentrations of inflammatory parameters which are subject to diurnal changes. We included 34 untreated patients with relapsing-remitting MS and 34 age- and sex-matched healthy controls. 12 MS patients showed acute disease activity in corresponding MRI scans. Blood samples were obtained at 7.00, 11.00 am, 2.30, 6.00 and 9.30 pm within 1 day. We determined serum levels of cortisol and inflammatory markers including soluble tumor necrosis factor-beta (sTNF-ß), soluble TNF-Receptor-1 (sTNF-R1) and -2 (sTNF-2), soluble vascular adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) by ELISA. We observed significantly higher serum levels of sTNF-R1 (p < 0.001) and sTNF-R2 (p < 0.001) in the morning and a significant decline of sICAM-1 (p < 0.005) and sVCAM-1 (p < 0.001) in the afternoon in both, MS patients and healthy controls. Comparison of diurnal serum levels between MS patients with active versus with non-active disease revealed significantly higher serum levels of sVCAM-1 (p < 0.05) around noon and in the early afternoon in MS patients with active disease. A significant decline of sICAM-1 (p < 0.05) in the afternoon was seen in MS patients with active and non-active disease. Our data indicate that increased awareness of potential diurnal serum concentration changes of biomarkers can eliminate one major cause of biased data as they occur in most of the investigated immunological parameters.