The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. β-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.
Background:CYP1A1, a member of the cytochrome P450 (CYP) enzymes, plays a very important role in metabolizing carcinogens. The aim of this case-control study was to detect the frequency of CYP1A1*2C polymorphism in Iranian leukemic patients and determine the role of this allele's variants, if any, as a risk factor for developing leukemia.
Genetic variations and mutations are the etiological factors of leukemia. NAD(P)H:quinone oxidoreductase (NQO1) plays an important role in the detoxification of quinones. C609T and C465T are 2 common polymorphisms in NQO1 resulting in lower NQO1 activity compared with wild type (CC). We assessed the frequency of C609T (NQO1*2; Proline to Serine) and C465T (NQO1*3; Arginine to Tryptophane) polymorphisms of the NQO1 gene among the Iranian population to determine the association between these polymorphisms and a susceptibility to adult acute myeloid leukemia (AML). Frequencies of NQO1 gene polymorphisms were determined in 140 AML patients for NQO1*2 and NQO1*3. In addition, 160 age-sex matched healthy individuals participated in this study as a control group. Genotyping was done using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assays. No significant association was observed between these 2 polymorphisms of NQO1 and the risk of AML. Odds ratio (OR) for C609T and C465T were 0.917 (95% CI=0.513–1.639) and 1.976 (95% CI=0.549–7.121), respectively. Men showed a higher incidence of C609T and C465T NQO1 than women. The majority of patients with a mutant allele were diagnosed as M3 sub type of French-American-British (FAB) classification. Our findings suggest that the NQO1 C609T and C465T gene variants do not have a major influence on the susceptibility to adult AML. Interestingly, we found a higher incidence of the T allele in NQO1*2 than NQO1*3 in the control and patient groups. Further studies are required to validate these findings across different populations.
Objectives: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme that protects cells against mutagenicity of free radicals and toxic oxygen metabolites. C to T base substitution at nucleotides 609 and 465 of NQO1 cDNA, results in loss of enzyme activity. Low NQO1 activity may play a role in etiology of ALL. In the present study, we investigated the association between the NQO1 polymorphisms and increased risk of ALL in children. Methods: C609T and C465T polymorphisms of NQO1 were explored using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assay in 100 pediatric ALL patients and 135 healthy controls. Results: Although C609T polymorphism is very common among the Iranian population, we found no association between this variant and increased risk for pediatric ALL [odds ratio (OR) = 0.95; 95% confidence interval (95% CI) = 0.55–1.64]. Interestingly the other polymorphic allele of NQO1 (C465T) was strongly associated with pediatric ALL (OR = 7.83; 95% CI= 3.27-18.75). Conclusion: These findings do not support the predisposing role of NQO1 C609T polymorphism for pediatric ALL. However, The C465T polymorphism was associated with increased risk of pediatric ALL. Further studies with larger sample including evaluating multiple gene– gene interactions seem necessary to validate the exact role of these mutations.
Zebrafish have proven
to be an excellent model for studying host-pathogen interactions. They share
highly conserved genetic and physiological aspects with mammals, and offer
technical advantages like small size, high fecundity, availability of several
genomic tools and the feasibility of large-scale phenotype screening.
I have used reverse genetic approaches to exploit the
zebrafish embryo model to study the molecular basis of severe congenital
neutropenia (SCN), a primary immunodeficiency, which is associated with various
severe lethal infections in early childhood. Using exome sequencing on known
families of SCN combined with linkage analysis and mapping approaches, a list
of candidate genes for SCN was provided by Christoph Klein, (Ludwig-Maximilians
University, Munich, Germany) that were potentially responsible for neutropenia
in SCN patients. A morpholino knockdown strategy was chosen as the initial
reverse genetic approach to see if loss of function of the smarcd2, a gene
prioritized for study by Dr Klein, could replicate the neutropenia in a
zebrafish model. The smarcd2 morphant embryos replicated the neutropenia
phenotype. A knockout model of Smarcd2 was pursued to further evaluate the role
of this gene in granulopoiesis, which provided a stable mutant line with a mild
neutrophil-deficiency phenotype. These studies confirmed a genetic requirement
for smarcd2 for neutrophil development in zebrafish in vivo.
Secondly, to understand the mechanisms by which the immune
system protects the host against pathogens, I have exploited zebrafish as an
infection model to see the interactions of immune elements with the fungal
opportunistic pathogen Penicillium marneffei. In addition to characterizing
macrophage dynamics during the course of infection in this model, I collected
multiple additional examples of shuttling of phagocytosed spores from
neutrophil to macrophage. This novel phenomenon had been previously
demonstrated in Lieschke lab, but only by four examples. With multiple
examples, I was able to quantify some aspects of this previously undescribed
phenomenon. By demonstrating shuttling of Aspergillus fumigatus spores, another
opportunistic fungi, I showed that shuttling is not a P. marneffei-specific
phenomenon. Having multiple examples of shuttling events with different
pathogens enabled this phenomenon to be characterised in more detail and also
provided more clues regarding a possible mechanism of spore shuttling. I showed
that polystyrene beads mimicking fungal spores are not shuttled. However,
zymosan particles, which are fungal cell wall derivatives, are shuttled. These
results provide a strong clue that zymosan contains the triggering factor for
shuttling and that it is a structural components in fungal cell wall, most
likely β-glucan.
Finally I evaluated the potential of this zebrafish embryo
fungal infection model to test novel therapeutic strategies against fungal infection.
I have provided proof-of-principle that it can be used as a tool for antifungal
therapeutic studies. I showed that the biology of macrophages could be
exploited to deliver antifungal drugs by showing that spore-laden macrophages
are capable of engulfing nanoparticles avidly. This makes it possible to
deliver antifungal agents (drugs, myeloperoxidase, etc.) inside macrophages,
which is where the P. marneffei spores hide themselves and survive other
effective immune elements.
The studies described in this thesis therefore provide new
discoveries about: (1) a new genetic cause of severe congenital neutropenia;
(2) neutrophil-to-macrophage pathogen shuttling, a novel host/pathogen
interaction; (3) the utility of zebrafish fungal infection models for exploring
antifungal therapeutic strategies.
Abstract Telomere biology disorders (TBDs) are inherited conditions associated with multisystem manifestations. We describe clinical and functional characterisation of a novel TERT variant. Whole‐genome sequencing was performed along with single telomere length analysis ( STELA ). Telomerase activity and processivity were assessed. A novel TERT variant (K710R) was detected in a patient with classic TBD features showing reduced telomerase activity and processivity. Despite clinical and functional evidence, the variant was classified as a variant of uncertain significance. We have described a novel TERT variant and highlighted the need for further refinement of variant classification specific for TBDs.
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