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1.
Nat Immunol ; 23(2): 330-340, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-35087231

RESUMEN

Intravital confocal microscopy and two-photon microscopy are powerful tools to explore the dynamic behavior of immune cells in mouse lymph nodes (LNs), with penetration depth of ~100 and ~300 µm, respectively. Here, we used intravital three-photon microscopy to visualize the popliteal LN through its entire depth (600-900 µm). We determined the laser average power and pulse energy that caused measurable perturbation in lymphocyte migration. Long-wavelength three-photon imaging within permissible parameters was able to image the entire LN vasculature in vivo and measure CD8+ T cells and CD4+ T cell motility in the T cell zone over the entire depth of the LN. We observed that the motility of naive CD4+ T cells in the T cell zone during lipopolysaccharide-induced inflammation was dependent on depth. As such, intravital three-photon microscopy had the potential to examine immune cell behavior in the deeper regions of the LN in vivo.


Asunto(s)
Microscopía Intravital/métodos , Ganglios Linfáticos/citología , Microscopía Confocal/métodos , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Movimiento Celular/fisiología , Rastreo Celular/métodos , Ratones
2.
Nat Mater ; 22(4): 511-523, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36928381

RESUMEN

Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly. We demonstrate that Ly-TME cellular and biophysical factors amplify the BCR-MYD88-TLR9 multiprotein supercomplex and induce cooperative signalling pathways in ABC-DLBCL cells, which reduce the efficacy of compounds targeting the BCR pathway members Bruton tyrosine kinase and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). Combinatorial inhibition of multiple aberrant signalling pathways induced higher antitumour efficacy in lymphoid organoids and implanted ABC-DLBCL patient tumours in vivo. Our studies define the complex crosstalk between malignant ABC-DLBCL cells and Ly-TME, and provide rational combinatorial therapies that rescue Ly-TME-mediated attenuation of treatment response to MALT1 inhibitors.


Asunto(s)
Linfoma de Células B Grandes Difuso , Microambiente Tumoral , Humanos , Línea Celular Tumoral , Transducción de Señal , FN-kappa B/metabolismo , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo
3.
Adv Funct Mater ; 30(48)2020 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-33692664

RESUMEN

Antibiotic-resistant bacteria are a major global health threat that continues to rise due to a lack of effective vaccines. Of concern are Klebsiella pneumoniae that fail to induce in vivo germinal center B cell responses, which facilitate antibody production to fight infection. Immunotherapies using antibodies targeting antibiotic-resistant bacteria are emerging as promising alternatives, however, they cannot be efficiently derived ex vivo, necessitating the need for immune technologies to develop therapeutics. Here, PEG-based immune organoids were developed to elucidate the effects of polymer end-point chemistry, integrin ligands, and mode of K. pneumoniae antigen presentation on germinal center-like B cell phenotype and epigenetics, to better define the lymph node microenvironment factors regulating ex vivo germinal center dynamics. Notably, PEG vinyl sulfone or acrylate failed to sustain primary immune cells, but functionalization with maleimide (PEG-4MAL) led to B cell expansion and germinal center-like induction. RNA sequencing analysis of lymph node stromal and germinal center B cells showed niche associated heterogeneity of integrin-related genes. Incorporation of niche-mimicking peptides revealed that collagen-1 promoted germinal center-like dynamics and epigenetics. PEG-4MAL organoids elucidated the impact of K. pneumoniae outer membrane-embedded protein antigen versus soluble antigen presentation on germinal centers and preserved the response across young and aged mice.

4.
Anal Chem ; 89(3): 1684-1688, 2017 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-28208284

RESUMEN

Papaverine is a non-narcotic alkaloid found endemically and uniquely in the latex of the opium poppy. It is normally refined out of the opioids that the latex is typically collected for, hence its presence in a sample is strong prima facie evidence that the carrier from whom the sample was collected is implicated in the mass cultivation of poppies or the collection and handling of their latex. We describe an analysis technique combining surface-enhanced Raman spectroscopy (SERS) with microfluidics for detecting papaverine at low concentrations and show that its SERS spectrum has unique spectroscopic features that allows its detection at low concentrations among typical opioids. The analysis requires approximately 2.5 min from sample loading to results, which is compatible with field use. The weak acid properties of papaverine hydrochloride were investigated, and Raman bands belonging to the protonated and unprotonated forms of the isoquinoline ring of papaverine were identified.

5.
ACS Cent Sci ; 9(4): 787-804, 2023 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-37122450

RESUMEN

Glycoengineered bacteria have emerged as a cost-effective platform for rapid and controllable biosynthesis of designer conjugate vaccines. However, little is known about the engagement of such conjugates with naïve B cells to induce the formation of germinal centers (GC), a subanatomical microenvironment that converts naïve B cells into antibody-secreting plasma cells. Using a three-dimensional biomaterials-based B-cell follicular organoid system, we demonstrate that conjugates triggered robust expression of hallmark GC markers, B cell receptor clustering, intracellular signaling, and somatic hypermutation. These responses depended on the relative immunogenicity of the conjugate and correlated with the humoral response in vivo. The occurrence of these mechanisms was exploited for the discovery of high-affinity antibodies against components of the conjugate on a time scale that was significantly shorter than for typical animal immunization-based workflows. Collectively, these findings highlight the potential of synthetic organoids for rapidly predicting conjugate vaccine efficacy as well as expediting antigen-specific antibody discovery.

6.
Sci Adv ; 5(3): eaav9788, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30944865

RESUMEN

Biomaterials-based nanovaccines, such as those made of poly(lactic-co-glycolic acid) (PLGA), can induce stronger immunity than soluble antigens in healthy wild-type mouse models. However, whether metabolic syndrome can influence the immunological responses of nanovaccines remains poorly understood. Here, we first show that alteration in the sensing of the gut microbiome through Toll-like receptor 5 (TLR5) and the resulting metabolic syndrome in TLR5 -/- mice diminish the germinal center immune response induced by PLGA nanovaccines. The PLGA nanovaccines, unexpectedly, further changed gut microbiota. By chronically treating mice with antibiotics, we show that disrupting gut microbiome leads to poor vaccine response in an obesity-independent manner. We next demonstrate that the low immune response can be rescued by an immunomodulatory Pyr-pHEMA nanogel vaccine, which functions through TLR2 stimulation, enhanced trafficking, and induced stronger germinal center response than alum-supplemented PLGA nanovaccines. The study highlights the potential for immunomodulation under gut-mediated metabolic syndrome conditions using advanced nanomaterials.


Asunto(s)
Modelos Animales de Enfermedad , Microbioma Gastrointestinal/efectos de los fármacos , Inmunidad/efectos de los fármacos , Síndrome Metabólico/prevención & control , Nanogeles , Polietilenglicoles , Polietileneimina , Vacunas/administración & dosificación , Análisis de Varianza , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Materiales Biocompatibles/administración & dosificación , Microbioma Gastrointestinal/inmunología , Inmunidad/inmunología , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/inmunología , Síndrome Metabólico/inmunología , Ratones Noqueados , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/inmunología , Receptor Toll-Like 5/metabolismo , Vacunas/inmunología
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