The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells.

Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages.

Use of autoantigen-loaded phosphatidylserine-liposomes to arrest autoimmunity in type 1 diabetes.

INTRODUCTION: The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow ß-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. OBJECTIVE: To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to ß-cells in type 1 diabetes. METHODS: A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. RESULTS: We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4+ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4+ T cell expansion. CONCLUSIONS: We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases.

Immunotherapy with Tolerogenic Dendritic Cells Alone or in Combination with Rapamycin Does Not Reverse Diabetes in NOD Mice.

Type 1 diabetes is a metabolic disease caused by autoimmunity towards ß -cells. Different strategies have been developed to restore ß -cell function and to reestablish immune tolerance to prevent and cure the disease. Currently, there is no effective treatment strategy to restore endogenous insulin secretion in patients with type 1 diabetes. This study aims to restore insulin secretion in diabetic mice with experimental antigen-specific immunotherapy alone or in combination with rapamycin, a compound well known for its immunomodulatory effect. Nonobese diabetic (NOD) mice develop spontaneous type 1 diabetes after 12 weeks of age. Autologous tolerogenic dendritic cells-consisting in dendritic cells pulsed with islet apoptotic cells-were administered to diabetic NOD mice alone or in combination with rapamycin. The ability of this therapy to revert type 1 diabetes was determined by assessing the insulitis score and by measuring both blood glucose levels and C-peptide concentration. Our findings indicate that tolerogenic dendritic cells alone or in combination with rapamycin do not ameliorate diabetes in NOD mice. These results suggest that alternative strategies may be considered for the cure of type 1 diabetes.

PCR-based microsatellite analysis to accelerate diabetogenic genetic background acquisition in transgenic mice

The Non-obese diabetic (NOD) mice exhibit a susceptibility to spontaneous development of autoimmune diabetes and is the most widely used experimental model for the study of the disease. The NOD strain was established by inbreeding in 1980. This model has a MHC-matched diabetes resistant homologous, NOR/Lt mice, an insulitis-resistant and diabetes-free strain produced from an isolated genetic contamination within a NOD/Lt line. To evaluate the role of transgenes, transgenic mice can be generated in CD-1 mice for technical advantages and then backcrossed to inbred strains. To obtain transgenic mice in NOD or NOR background starting from CD-1, at least 20 backcrosses are required, spending more than two years in the process.Nucleotide repeats (microsatellites) mapped to specific locations on each chromosome are used to evaluate genomic polymorphism. From 23 microsatellites we selected eleven that were variant in PCR amplimer size between CD-1 colony and NOD or NOR strains. We used these microsatellites to identify individuals that were used for backcrossing, thus accelerating the acquisition of a new genetic background. Results yield a defined analysis of the genome in question and profiles were compared to detect genetic variation among individuals. After the selection of mice for backcrossing at the third generation, the 11 specific markers were acquired at the 5th generation and maintained to the 10th generation. Diabetes incidence and insulitis score correlated with the acquisition of genetic background, demonstrating that using this strategy, 5-6 crosses are enough to obtain the genotype of interest, shortening the process in more than one year and a half (AU)

Los ratones diabéticos no obesos (NOD) presentan susceptibilidad al desarrollo espontáneo de diabetes autoinmunitaria y son el modelo experimental más utilizado para el estudio de la enfermedad. La cepa NOD fue establecida por endogamia en 1980. Este modelo tiene un homólogo resistente a la diabetes con MHC compatible, el ratón NOR/Lt, una cepa sin diabetes y que no desarrolla insulitis producida a partir de una contaminación genética aislada en una línea de NOD/Lt. Para evaluar la función de los transgenes, los ratones transgénicos pueden generarse en ratones CD1 por sus ventajas técnicas y luego cruzarse con cepas consanguíneas. Para obtener ratones transgénicos con genotipo NOD o NOR a partir de CD1, se requieren como mínimo 20 retrocruzamientos, lo que supone un proceso de más de dos años.Las repeticiones de nucleótidos (microsatélites) identificadas en localizaciones específicas de cada cromosoma se utilizan para evaluar el polimorfismo genómico. De 23 microsatélites, seleccionamos 11 con tamaños de amplímero de PCR divergentes entre la colonia de CD1 y las cepas NOD o NOR. Utilizamos estos microsatélites para identificar individuos que se utilizaron para los retrocruzamientos, con lo que se aceleró la adquisición de unos nuevos genotipos. Los resultados produjeron un análisis definido del genoma en cuestión y se compararon los perfiles para detectar variaciones genéticas entre los individuos. Tras la selección de ratones para llevar a cabo el retrocruzamiento en la tercera generación, los 11 marcadores específicos se adquirieron en la 5.ª generación y se mantuvieron hasta la 10.ª generación. La puntuación de insulitis e incidencia de diabetes se correlacionó con la adquisición de genotipos, lo que demostró que mediante esta estrategia bastaban 5-6 retrocruzamientos para obtener el genotipo de interés, reduciendo así el proceso en más de un año y medio (AU)