Monoclonal Antibodies against Cell Wall Chitooligomers as Accessory Tools for the Control of Cryptococcosis.

Therapeutic strategies against systemic mycoses can involve antifungal resistance and significant toxicity. Thus, novel therapeutic approaches to fight fungal infections are urgent. Monoclonal antibodies (MAbs) are promising tools to fight systemic mycoses. In this study, MAbs of the IgM isotype were developed against chitin oligomers. Chitooligomers derive from chitin, an essential component of the fungal cell wall and a promising therapeutic target, as it is not synthesized by humans or animals. Surface plasmon resonance (SPR) assays and cell-binding tests showed that the MAbs recognizing chitooligomers have high affinity and specificity for the chitin derivatives. In vitro tests showed that the chitooligomer MAbs increased the fungicidal capacity of amphotericin B against Cryptococcus neoformans. The chitooligomer-binding MAbs interfered with two essential properties related to cryptococcal pathogenesis: biofilm formation and melanin production. In a murine model of C. neoformans infection, the combined administration of the chitooligomer-binding MAb and subinhibitory doses of amphotericin B promoted disease control. The data obtained in this study support the hypothesis that chitooligomer antibodies have great potential as accessory tools in the control of cryptococcosis.

Unleashing Fungicidal Forces: Exploring the Synergistic Power of Amphotericin B-Loaded Nanoparticles and Monoclonal Antibodies.

Fungal infections cause 1.7 million deaths annually, which can be attributed not only to fungus-specific factors, such as antifungal resistance and biofilm formation, but also to drug-related challenges. In this study, the potential of Amphotericin (AmB) loaded polymeric nanoparticles (AmB-NPs) combined with murine monoclonal antibodies (mAbs) (i.e., CC5 and DD11) was investigated as a strategy to overcome these challenges. To achieve this goal, AmB-NPs were prepared by nanoprecipitation using different polymers (polycaprolactone (PCL) and poly(D,L-lactide) (PLA)), followed by comprehensive characterization of their physicochemical properties and in vitro biological performance. The results revealed that AmB-loaded NPs exhibited no cytotoxicity toward mammalian cells (baby hamster kidney cells-BHK and human monocyte cells-THP-1). Conversely, both AmB-NPs demonstrated a cytotoxic effect against C. albicans, C. neoformans, and H. capsulatum throughout the entire evaluated range (from 10 µg/mL to 0.1 µg/mL), with a significant MIC of up to 0.031 µg/mL. Moreover, the combination of AmB-NPs with mAbs markedly intensified antifungal activity, resulting in a synergistic effect that was two to four times greater than that of AmB-NPs alone. These findings suggest that the combination of AmB-NPs with mAbs could be a promising new treatment for fungal infections that is potentially more effective and less toxic than current antifungal treatments.

Gedunin, a natural tetranortriterpenoid, modulates T lymphocyte responses and ameliorates allergic inflammation.

T lymphocytes are critical cells involved in allergy. Here, we report that the natural tetranortriterpenoid gedunin impaired allergic responses primarily by modulating T lymphocyte functions. The intraperitoneal (i.p.) administration of gedunin inhibited pleural leukocyte accumulation triggered by intra-pleural (i.pl.) challenge with ovalbumin (OVA) in previously sensitized C57BL/6 mice; this inhibition was primarily due to the impairment of eosinophil and T lymphocyte influx. Likewise, i.pl. pre-treatment with gedunin inhibited eosinophil and T lymphocyte migration into mouse lungs 24 h after OVA intra-nasal (i.n.) instillation. Pre-treatment with gedunin diminished the levels of CCL2, CCL3, CCL5, CCL11, Interleukin-5 and leukotriene B(4) at the allergic site. In vitro pre-treatment with gedunin failed to inhibit T lymphocyte adhesion and chemotaxis towards pleural washes recovered from OVA-challenged mice, suggesting that gedunin inhibits T lymphocyte migration in vivo via the inhibition of chemotactic mediators in situ. In vivo pre-treatment with gedunin reduced the numbers of CD69(+) and CD25(+) T lymphocytes in the pleura and CD25(+) cells in the thoracic lymph nodes 24 h after OVA i.pl. challenge. In accordance, in vitro treatment of T lymphocytes with gedunin inhibited α-CD3 mAb-induced expression of CD69 and CD25, proliferation, Interleukin-2 production and nuclear translocation of NFκB and NFAT. Notably, post-treatment of mice with gedunin reverted OVA-induced lung allergic inflammation by decreasing the T lymphocyte and eosinophil counts and the levels of eosinophilotactic mediators in bronchoalveolar lavage fluid. Our results demonstrate a remarkable anti-allergic effect of gedunin due to its capability to modulate T cell activation and trafficking into the airways.

Rastreamento de mutações no gene GATA1 em crianças com síndrome de Down

Crianças com síndrome de Down (SD) apresentam um risco 10 a 20 vezes maior de desenvolver leucemia do que crianças normais, particularmente a leucemia megacarioblástica aguda (LMA-M7) e uma forma reversível denominada doença mieloproliferativa transitória também conhecida como leucemia transitória (LT), devido ao fato de que geralmente há uma remissão espontânea dentro de 3 meses. A LT pode ser considerada uma pré-condição leucêmica, já que cerca de 20% dos pacientes podem desenvolver a LMA-M7 no prazo de 4 anos. Recentemente, foi relatado que mutações somáticas no GATA1, localizado no cromossomo X, estão presentes tanto em blastos de LT quanto em LMA-M7 de crianças com SD. O GATA1 é um fator de transcrição e está presente na diferenciação normal das linhagens eritróides e megacariocíticas. O modo pelo qual as alterações no GATA1 contribuem para a leucemia ainda é desconhecido. A partir disso, estabelecemos um programa nacional, a fim de determinar a incidência de mutações no GATA1 (éxons 2 e 3) em uma coorte de recém-nascidos com SD. Para isso, utilizamos a técnica de cromatografia líquida desnaturante de alta performance (dHPLC) e seqüenciamento automático. Esta técnica de dHPLC se baseia nas variações de heteroduplex e homoduplex dos fragmentos de DNA e apesar de o seqüenciamento automático ser o padrão ouro para a identificação de mutações, este método pode ser lento quanto à análise da mutação, ao passo que o dHPLC tem se mostrado eficaz e rápido para a análise das variações genéticas de diversos genes de interesse médico. Para este estudo utilizamos medula óssea e/ou sangue periférico de 111 crianças com SD (recém-nascidos e crianças sendo a grande maioria com menos de 4 anos de idade) obtidos entre janeiro de 2000 e dezembro de 2007, sem tratamento prévio. Um total de 127 amostras de crianças com SD foram analisadas, sendo 66 crianças com SD e doenças hematológicas identificadas clinicamente e 61 recémnascidos com SD e sem evidência clínica de doenças hematológicas. A análise através do dHPLC e seqüenciamento automático identificou dezenove mutações no éxon 2 exclusivamente em crianças com LT e LMA-M7 com SD e em uma criança com LT e SD foi detectada alteração no éxon 3.A freqüência de anomalias genéticas não foi estatisticamente significativa em relação ao sexo ou cor da pele e alterações no GATA1 não foram detectadas em nossa coorte de recém-nascidos sem sinal de distúrbios hematológicos. A concordância da detecção através da técnica de dHPLC foi de 100% com o seqüenciamento automático. Em conclusão, nossos resultados indicam que alterações no GATA1 são especificas do subtipo LMA-M7 e LT da SD e que a técnica de dHPLC é eficaz e uma valiosa ferramenta para análise mutacional no GATA1 e, além disso, podemos consolidar o GATA1 como um marcador molecular com o intuito de uniformizar os critérios diagnósticos precoces da criança com SD melhorando assim sua taxa de sobrevida.

Children with Down syndrome (DS) have a 10 to 20-fold elevated risk of developing leukaemia, particularly acute megakaryoblastic leukemia (AMKL) and a reversible form of myeloproliferative disorder, known as transient leukemia (TL), which usually spontaneous resolves within 3 months. TL can be considered a preleukemic condition, as approximately 20% of TL patients will develop AMKL within 4 years. Recently, it has been reported that somatic mutations in the X-linked GATA1 gene are present in TL and AMKL blasts of DS infants. GATA1 gene encodes a transcription factor that is critical for normal development of erythroid and megakaryocytic lineages. The precise pathway by which mutagenesis of GATA1 contributes to leukemia is still unknown. Then, we established a national program in order to determine the incidence of GATA1 mutations in a cohort of DS newborns and children with DS presenting hematological disorders, furthermore we have evaluated the efficacy of denaturing high-performance liquid chromatography (dHPLC) screening method for detecting mutations in GATA1 gene. Bone marrow and/or peripheral blood from 111 DS children (newborns and children with the vast majority less than 4 years old) obtained between January 2000 and December 2007 without previous treatment. They were screened for GATA1 mutations (exons 2 e 3) by the denaturing High-Performance Liquid Chromatographic (dHPLC) and direct sequencing in an automated sequencer. dHPLC has been developed to screen for DNA variations by separating heteroduplex and homoduplex DNA fragments by ion-pair reverse-phase liquid chromatography. Although the automatic sequencing is the gold standard technique for identifying mutations, this method can be time consuming for analysis, while the dHPLC was effective and fast for the analysis of genetic variations A total of 127 samples from DS children were analyzed, with 66 DS children with hematological disorders identified clinically and 61 newborns without clinical evidence of hematological disorders by dHPLC and direct sequencing methods. Nineteen mutations were detected exclusively in exon 2 of DS children with AMKL nd TL disorders and one was detected in exon 3 of DS child with TL. The frequency of genetic abnormalities was no statistically significant regarding to sex or ethnicityand GATA 1 mutation was not detected in our cohort of newborns without sign of hematological disorder. The overall detection rate of dHPLC screening was 100%. In conclusion, our results indicate that dHPLC is an efficient and valuable tool for GATA1 mutational analysis.