A Bacterial and Ganglioside-based Nanoparticle Initiates Reprogramming of Macrophages and Promotes Antitumor Phenotypes.

Macrophages represent the most abundant immune component of the tumor microenvironment and often exhibit protumorigenic (M2-like) phenotypes that contribute to disease progression. Despite their generally accepted protumorigenic role, macrophages can also display tumoricidal (or M1-like) behavior, revealing that macrophages can be functionally reprogrammed, depending on the cues received within the tumor microenvironment. Moreover, such plasticity may be achieved by pharmacologic or biologic interventions. To that end, we previously demonstrated that a novel immunomodulator termed the "very small size particle" (VSSP) facilitates maturation of dendritic cells and differentiation of myeloid-derived suppressor cells to APCs with reduced suppressive activity in cancer models. VSSP was further shown to act in the bone marrow to drive the differentiation of progenitors toward monocytes, macrophages, and dendritic cells during emergency myelopoiesis. However, the underlying mechanisms for VSSP-driven alterations in myeloid differentiation and function remained unclear. In this study, in mouse models, we focused on macrophages and tested the hypothesis that VSSP drives macrophages toward M1-like functional states via IRF8- and PU.1-dependent mechanisms. We further hypothesized that such VSSP-mediated actions would be accompanied by enhanced antitumor responses. Overall, we showed that (1) VSSP drives naive or M2-derived macrophages to M1-like states, (2) the M1-like state induced by VSSP occurs via IRF8- and PU.1-dependent mechanisms, and (3) single-agent VSSP induces an antitumor response that is accompanied by alterations in the intratumoral myeloid compartment. These results provide a deeper mechanistic underpinning of VSSP and strengthen its use to drive M1-like responses in host defense, including cancer.

Novel Adjuvant Based on the Pore-Forming Protein Sticholysin II Encapsulated into Liposomes Effectively Enhances the Antigen-Specific CTL-Mediated Immune Response.

Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immunomodulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response.

Inhibition of tumor-induced myeloid-derived suppressor cell function by a nanoparticulated adjuvant.

The interaction between cancer vaccine adjuvants and myeloid-derived suppressor cells (MDSCs) is currently poorly understood. Very small size proteoliposomes (VSSP) are a nanoparticulated adjuvant under investigation in clinical trials in patients with renal carcinoma, breast cancer, prostate cancer, and cervical intraepithelial neoplasia grade III. We found that VSSP adjuvant induced a significant splenomegaly due to accumulation of CD11b(+)Gr-1(+) cells. However, VSSP-derived MDSCs showed a reduced capacity to suppress both allogeneic and Ag-specific CTL response compared with that of tumor-induced MDSCs. Moreover, splenic MDSCs isolated from tumor-bearing mice treated with VSSP were phenotypically more similar to those isolated from VSSP-treated tumor-free mice and much less suppressive than tumor-induced MDSCs, both in vitro and in vivo. Furthermore, different from dendritic cell vaccination, inoculation of VSSP-based vaccine in EG.7-OVA tumor-bearing mice was sufficient to avoid tumor-induced tolerance and stimulate an immune response against OVA Ag, similar to that observed in tumor-free mice. This effect correlated with an accelerated differentiation of MDSCs into mature APCs that was promoted by VSSP. VSSP used as a cancer vaccine adjuvant might thus improve antitumor efficacy not only by stimulating a potent immune response against tumor Ags but also by reducing tumor-induced immunosuppression.

Mitigating the prevalence and function of myeloid-derived suppressor cells by redirecting myeloid differentiation using a novel immune modulator.

BACKGROUND: Immune suppression is common in neoplasia and a major driver is tumor-induced myeloid dysfunction. Yet, overcoming such myeloid cell defects remains an untapped strategy to reverse suppression and improve host defense. Exposure of bone marrow progenitors to heightened levels of myeloid growth factors in cancer or following certain systemic treatments promote abnormal myelopoiesis characterized by the production of myeloid-derived suppressor cells (MDSCs) and a deficiency in antigen-presenting cell function. We previously showed that a novel immune modulator, termed 'very small size particle' (VSSP), attenuates MDSC function in tumor-bearing mice, which was accompanied by an increase in dendritic cells (DCs) suggesting that VSSP exhibits myeloid differentiating properties. Therefore, here, we addressed two unresolved aspects of the mechanism of action of this unique immunomodulatory agent: (1) does VSSP alter myelopoiesis in the bone marrow to redirect MDSC differentiation toward a monocyte/macrophage or DC fate? and (2) does VSSP mitigate the frequency and suppressive function of human tumor-induced MDSCs? METHODS: To address the first question, we first used a murine model of granulocyte-colony stimulating factor-driven emergency myelopoiesis following chemotherapy-induced myeloablation, which skews myeloid output toward MDSCs, especially the polymorphonuclear (PMN)-MDSC subset. Following VSSP treatment, progenitors and their myeloid progeny were analyzed by immunophenotyping and MDSC function was evaluated by suppression assays. To strengthen rigor, we validated our findings in tumor-bearing mouse models. To address the second question, we conducted a clinical trial in patients with metastatic renal cell carcinoma, wherein 15 patients were treated with VSSP. Endpoints in this study included safety and impact on PMN-MDSC frequency and function. RESULTS: We demonstrated that VSSP diminished PMN-MDSCs by shunting granulocyte-monocyte progenitor differentiation toward monocytes/macrophages and DCs with heightened expression of the myeloid-dependent transcription factors interferon regulatory factor-8 and PU.1. This skewing was at the expense of expansion of granulocytic progenitors and rendered the remaining MDSCs less suppressive. Importantly, these effects were also demonstrated in a clinical setting wherein VSSP monotherapy significantly reduced circulating PMN-MDSCs, and their suppressive function. CONCLUSIONS: Altogether, these data revealed VSSP as a novel regulator of myeloid biology that mitigates MDSCs in cancer patients and reinstates a more normal myeloid phenotype that potentially favors immune activation over immune suppression.

Cancer-induced systemic myeloid dysfunction: Implications for treatment and a novel nanoparticle approach for its correction.

Unlike other regulatory circuits, cancer-induced myeloid dysfunction involves more than an accumulation of impaired dendritic cells, protumoral macrophages, and myeloid derived suppressor cells in the tumor microenvironment. It is also characterized by "aberrant" myelopoiesis that results in the accumulation and expansion of immature myeloid precursors with a suppressive phenotype in the systemic circulation. The first part of this review briefly describes the evidence for and consequences of this systemic dysfunctional myelopoiesis and the possible reinforcement of this phenomenon by conventional treatments used in patients with cancer, in particular chemotherapy and granulocyte-colony stimulating factor. The second half of this review describes very small size particles, a novel immune-modulatory nanoparticle, and the evidence indicating a possible role of this agent in correcting or re-programming the dysfunctional myelopoiesis in different scenarios.

Determination of range of mandibular movements in children without temporomandibular disorders.

UNLABELLED: Mandibular movement values are an important parameter within the clinical evaluation of the temporomandibular joint. Limited or increased movement is a sign of dysfunction. Normal values used as reference correspond to adult populations, and information on child populations is scant. The aim of this study was to establish reference measurements of children with primary (Group A) and mixed dentition (Group B) without signs of temporomandibular disorders. The study population comprised 212 boys and girls, aged 3 to 11 years, attending a state school in the San Martin district in the province of Buenos Aires, who presented no joint sounds, clicking or pain. A calibrated operator determined maximal opening, protrusion, and lateral movements. Group A (n=105): mean age 4.61+/-0.9; maximal opening 38.59 mm +/- 4.03; protrusion 3.71 mm+/-1.79; right lateral movement 5.43 mm+/-1.83 and left lateral movement 5.52 mm +/- 1.73. Group B (n= 107): mean age 6.9+/-1.65; maximal opening 41.97 mm +/- 5.27; protrusion 3.96 mm+/-1.92; right lateral movement 6.05 mm+/-1.99 and left lateral movement 6.13 mm+/-2.21. Opening and lateral movements were found to increase with age. Comparison between groups using Welch t Test showed significant differences in maximal opening (p<0.0001), right (p= 0.0191) and left (p=0.0262) lateral movement. CONCLUSIONS: Mandibular movements are associated with growth. Mandibular movements of pediatric patients must be assessed in relation to age and type of dentition.

Adjuvants and myeloid-derived suppressor cells: enemies or allies in therapeutic cancer vaccination.

Adjuvants are a critical but largely overlooked and poorly understood component included in vaccine formulations to stimulate and modulate the desired immune responses to an antigen. However, unlike in the protective infectious disease vaccines, adjuvants for cancer vaccines also need to overcome the effect of tumor-induced suppressive immune populations circulating in tumor-bearing individuals. Myeloid-derived suppressor cells (MDSC) are considered to be one of the key immunosuppressive populations that inhibit tumor-specific T cell responses in cancer patients. This review focuses on the different signals for the activation of the immune system induced by adjuvants, and the close relationship to the mechanisms of recruitment and activation of MDSC. This work explores the possibility that a cancer vaccine adjuvant may either strengthen or weaken the effect of tumor-induced MDSC, and the crucial need to address this in present and future cancer vaccines.

Very small size proteoliposomes abrogate cross-presentation of tumor antigens by myeloid-derived suppressor cells and induce their differentiation to dendritic cells.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are among the major obstacles that adjuvants for cancer vaccines have to overcome. These cells cross-present tumor-associated antigens (TAA) to naive T lymphocytes with a tolerogenic outcome. Very Small Size Proteoliposomes (VSSP) is used as adjuvant by four therapeutic cancer vaccines currently in Phase I and II clinical trials. We previously found that VSSP reduces the suppressive function of MDSCs, then activating antigen-specific CTL responses in tumor-bearing (TB) mice, with the consequent reduction of tumor growth. However the mechanistic explanation for the immunomodulatory effect of this adjuvant in TB hosts has not been addressed before. METHODS: TB mice were inoculated with VSSP and MDSCs isolated and characterized by their expression of Arg1 and Nos2 genes by RT-PCR. The effect of VSSP on antigen cross-presentation by MDSCs, regulatory T cells (Tregs) expansion and MDSCs differentiation towards dendritic cells (DCs) was analyzed by FACS. Student's t test or ANOVA and Tukey's tests were used for statistical analyses. RESULTS: After inoculating VSSP into TB mice, a significant reduction of Arg1 and Nos2 gene expression was observed in recovered MDSCs. Concurrently the ability of these cells to induce down-regulation of CD3ζ chain on T cells was lost. Likewise in mice inoculated with the adjuvant lower percentages of Tregs were detected. In vitro, VSSP treatment was enough to differentiate MDSCs into phenotypically mature DCs, eliminating the former suppressive effect. Noteworthy, in vivo administration of VSSP to OVA-expressing (EG.7) TB mice abrogated this model antigen cross-presentation by splenic MDSCs. Similar results were obtained even when OVA antigen was administered into these TB mice formulated in VSSP. On the contrary, immunization with the same protein in polyI:C did not change the percentage of MDSCs expressing SIINFEKL/H-2K(b) complexes, whereas a concomitant injection of VSSP aborted the limitations of polyI:C in this setting. CONCLUSIONS: Altogether, these results indicate that VSSP has the peculiar capacity of inhibiting TAA cross-presentation and certain suppressive mechanisms on MDSCs which in turn, combined with the ability to induce differentiation of these cells into antigen-presenting cells (APCs), sustains this adjuvant as an ideal immunomodulator for cancer immunotherapy.

Very small size proteoliposomes derived from Neisseria meningitidis: an effective adjuvant for antigen-specific cytotoxic T lymphocyte response stimulation under leukopenic conditions.

Leukopenia is a severe condition resulting from both pathological processes and some treatments, like chemotherapy in cancer patients. However, the activation of the patient immune system is required for the success of immunotherapeutic strategies, as cancer vaccines. In this regard, leukopenia constitutes a major hurdle to overcome, mainly due to the impairment of cytotoxic T lymphocyte (CTL) responses. Adjuvants are basic components of vaccine formulations, which might be useful to stimulate immunity under this immunosuppressed condition. To this aim, we tested the capacity of a novel nanoparticulated complex, very small size proteoliposomes (VSSP), to promote CTL even in a leukopenic scenario. Noteworthy, we observed that a VSSP-based OVA vaccine induced a normal antigen-specific CTL response in mice rendered leukopenia by the administration of high doses of the chemotherapeutic agent cyclophosphamide (CY), while under the same conditions the OVA antigen formulated in the TLR-3 agonist polyinosinic-polycytidylic acid (P(I:C)) was ineffective. Moreover, an appropriate combination of VSSP with the P(I:C) vaccine was able to restore the CD8(+) T cell effector function in leukopenic mice. VSSP induced not only a faster repopulation of immune cells in CY-receiving animals, but also enhanced the recovery of memory T lymphocytes and myeloid dendritic cells (DCs) while simultaneously abrogated the immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs). Our results suggest that VSSP could be a particularly suitable immunomodulator to be used in CTL-promoting active immunotherapy strategies operating in severe immune compromised scenarios.

Desgaste incisal y oclusal como indicador de patología parafuncional en dentición primaria / Incisal and occlusal wear as indicators of parafunctional pathology in primary dentition

Un concepto tradicional en odontopediatría refiere el desgaste fisiológico de las piezas primarias. El objetivo de este trabajo fue identificar y cuantificar el desgaste incisal y oclusal ocasionado por bruxismo en piezas primarias de niños que concurren a escuelas públicas del Partido de Gral. San Martín, Buenos Aires. Material y método: se confeccionó una planilla de registro, con un cuestionario estructurado para padres, que identificó datos personales, alteraciones sistémicas, tipo y hábitos de alimentación, parasomnias, conducta, hábitos orales y bruxismo. Un docente calibrado realizó el registro dentario de desgaste categroizado de 0 a 3 (TWI). La muestra quedó constituida por 294 niños de ambos sexos, edad media 5,9 + - 1,9 años. Los valores de desgaste de cada pieza dentaria se integraron a una base de datos, dividiéndose en 2 grupos: bruxan (B) y no bruxan (NB), según las respuestas del cuestionario. Cada grupo se categorizó por edad y por serie dentaria (molares, caninos e incisivos). Resultados: se identificaron varios medios de desgaste para las series dentarias en las diferentes edades con y sin parafuncionales. Los subgrupos de B fueron comparados estadísticamente con los de NB. Se observaron diferencias significativas de desgaste (Chi cuadrado) para las distintas series dentarias a todas las edades. En B fue significativa (Fisher p < 0.0001) la presencia de niños ansiosos, sin hallazgos para el resto de las variables. Conclusiones: el desgaste dentario de piezas primarias debe considerarse en función de la edad y serie. La presencia de dentina expuesta a edades tempranas en esta muestra, podría considerarse como indicador de parafunción.

Comparación del estado bucal de niños con cardiopatías congénitas / A comparison of the oral status of children with congenital heart diseases

Las cardiopatías congénitas constituyen una de las más comunes alteraciones de desarrollo en niños y requieren atención odontológica debido a la susceptibilidad a desarrollar endocarditis bacteriana como consecuencia de infecciones bucales. El objetivo de este trabajo fue evaluar el estado bucal de niños con cardiopatías congénitas y compararlo con un grupo de niños sanos de la misma edad y sexo. Se evaluaron clínicamente 2 grupos de niños, uno portador de cardiopatías congénitas (GCC) y el otro sano (GC), registrando CPOS, ceos, defectos en el esmalte (índice DDE) y aspecto y coloración de tejidos blandos. GCC estuvo constituido por 38 niños entre 2 y 14 años portadores de cardiopatías congénitas, atendidos en el consultorio de mediano riesgo del Hospital Pedro Garrahan. GC se conformó seleccionando al azar un niño sano de la misma fecha de nacimiento y sexo por cada niño portador de cardiopatía congénita. Los datos fueron analizados estadísticamente por test de Welch. Al comparar ambos grupos no se hallaron diferencias significativas en CPOS (p=0.13) y ceos (P= 0.01) observándose hipoplasias de esmalte en el 26 por ciento de los pacientes de GCC. Conclusiones: los niños con cardiopatías congénitcas deben considerarse pacientes de alto riesgo cariogénico, siendo necesario implementar programas de educación para la salud para profesionales, padres y pacinetes, enfatizando la cooperación entre cardiólogos y odontopediatras

Factores asociados con bruxismo en niños y su prevalencia / Factors associated with bruxism in children and its prevalence

En la actualidad son muy frecuentes las consultas por bruxismo en la clínica odontopediátrica. Los factores vinculados con su etiología son múltiples, no endontrándose aún totalmente definidos, aunque se lo considera una parasomnia primaria no específica del sueño. POr ser una parafunción incociente, cuyos signos y síntomas no se observan en forma inmediata, el interrogatorio a padres y pacientes es el elemento más importante para el diagnóstico. El objetivo de este estudio es analizar la prevalencia de bruxismo en dentición primaria y mixta, y relacionarla con los factores más frecuentemente asociados. Material y métodos: se evaluaron 172 niños de ambos sexos de 5.61 + 0.48 años con dentición priamaria (DP) y 195 de 8.83 + 1-03 con dentición mixta (DM), pertenecientes a escuelas públicas de Gran Buenos Aires. Se utilizó un cuestionario domiciliario dirigido a los responsables del niño, donde se interrogó sexo, edad, rechinamiento, alteraciones del sueño, hábitos alimenticios, dolor de oídos y/o cabeza, respiración bucal y otros hábitos orales, parásitos y antecedentes familiares de bruxismo. Las opciones de respuesta fueron por sí o por no. Clínicamente se evaluó tipo de dentición, apertura máxima con y sin dolor, ruido y dolor articular, facetas de desgaste y alteraciones de la oclusión. Los datos fueron procesados en base de datos y analizados estadísticamente por Test t de Fisher. La prevalencia de bruxismo fue del 29 por ciento en dentición primaria y 17.94 por ciento en dentición mixta, vinculándose positivamente en ambas denticiones con otras alteraciones del sueño (DP p<0.0001, DM p=0.0015), respiración b ucal (DP p=0.0165, DM p=0.0018), dieta blanda (DP p<0.0001, DM p<0.0001) y dolor de oído y/o cabeza (DP p=0.0212, DM p=0.0321). Conclusiones: en esta población se observó mayor prevalencia de bruxismo en niños con dentición primaria, vinculándose en ambas denticiones con respiración bucal, otras alteraciones del sueño, dificultades en la alimentación y dolor de oídos y/o cabeza

Trastornos temporomandibulares en niños y adolescentes / Temporomandibular joint disorders in children and adolescents

Actualmente es frecuente que niños y adolescentes presenten bruxismo y trastornos temporomandibulares, los odontopediatras carecen de respuesta para estas patologías y parafunciones. El objetivo de este artículo es alertar sobre el problema, describir el examen clínico y radiográfico y presentar los distintos tratamientos desde un equipo interdisciplinario acorde al diagnóstico de pacientes en crecimiento y desarrollo