New Insights in Staging and Chemotherapy of African Trypanosomiasis and Possible Contribution of Medicinal Plants

Human African trypanosomiasis (HAT) is a fatal if untreated fly-borne neuroinflammatory disease caused by protozoa of the species Trypanosoma brucei (T.b.). The increasing trend of HAT cases has been reversed, but according to WHO experts, new epidemics of this disease could appear. In addition, HAT is still a considerable burden for life quality and economy in 36 sub-Saharan Africa countries with 15–20 million persons at risk. Following joined initiatives of WHO and private partners, the fight against HAT was re-engaged, resulting in considerable breakthrough. We present here what is known at this day about HAT etiology and pathogenesis and the new insights in the development of accurate tools and tests for disease staging and severity monitoring in the field. Also, we elaborate herein the promising progresses made in the development of less toxic and more efficient trypanocidal drugs including the potential of medicinal plants and related alternative drug therapies

Trypanosoma brucei brucei invasion and T-cell infiltration of the brain parenchyma in experimental sleeping sickness: timing and correlation with functional changes

Background: The timing of Trypanosoma brucei entry into the brain parenchyma to initiate the second, meningoencephalitic stage of human African trypanosomiasis or sleeping sickness is currently debated and even parasite invasion of the neuropil has been recently questioned. Furthermore, the relationship between neurological features and disease stage are unclear, despite the important diagnostic and therapeutic implications. Methodology: Using a rat model of chronic Trypanosoma brucei brucei infection we determined the timing of parasite and T-cell neuropil infiltration and its correlation with functional changes. Parasite DNA was detected using trypanosome-specific PCR. Body weight and sleep structure alterations represented by sleep-onset rapid eye movement (SOREM) periods, reported in human and experimental African trypanosomiasis, were monitored. The presence of parasites, as well as CD4+ and CD8+ T-cells in the neuropil was assessed over time in the brain of the same animals by immunocytochemistry and quantitative analyses. Principal findings: Trypanosome DNA was present in the brain at day 6 post-infection and increased more than 15-fold by day 21. Parasites and T-cells were observed in the parenchyma from day 9 onwards. Parasites traversing blood vessel walls were observed in the hypothalamus and other brain regions. Body weight gain was reduced from day 7 onwards. SOREM episodes started in most cases early after infection, with an increase in number and duration after parasite neuroinvasion. Conclusion: These findings demonstrate invasion of the neuropil over time, after an initial interval, by parasites and lymphocytes crossing the blood-brain barrier, and show that neurological features can precede this event. The data thus challenge the current clinical and cerebrospinal fluid criteria of disease staging

Expression of interferon-inducible chemokines and sleep/wake changes during early encephalitis in experimental African trypanosomiasis

Background: Human African trypanosomiasis or sleeping sickness, caused by the parasite Trypanosoma brucei, leads to neuroinflammation and characteristic sleep/wake alterations. The relationship between the onset of these alterations and the development of neuroinflammation is of high translational relevance, but remains unclear. This study investigates the expression of interferon (IFN)-γ and IFN-inducible chemokine genes in the brain, and the levels of CXCL10 in the serum and cerebrospinal fluid prior to and during the encephalitic stage of trypanosome infection, and correlates these with sleep/wake changes in a rat model of the disease. Methodology/Principal findings: The expression of genes encoding IFN-γ, CXCL9, CXCL10, and CXCL11 was assessed in the brain of rats infected with Trypanosoma brucei brucei and matched controls using semi-quantitative end-point RT-PCR. Levels of CXCL10 in the serum and cerebrospinal fluid were determined using ELISA. Sleep/wake states were monitored by telemetric recording. Using immunohistochemistry, parasites were found in the brain parenchyma at 14 days post-infection (dpi), but not at 6 dpi. Ifn-γ, Cxcl9, Cxcl10 and Cxcl11 mRNA levels showed moderate upregulation by 14 dpi followed by further increase between 14 and 21 dpi. CXCL10 concentration in the cerebrospinal fluid increased between 14 and 21 dpi, preceded by a rise in the serum CXCL10 level between 6 and 14 dpi. Sleep/wake pattern fragmentation was evident at 14 dpi, especially in the phase of wake predominance, with intrusion of sleep episodes into wakefulness. Conclusions/Significance: The results show a modest increase in Cxcl9 and Cxcl11 transcripts in the brain and the emergence of sleep/wake cycle fragmentation in the initial encephalitic stage, followed by increases in Ifn-γ and IFN-dependent chemokine transcripts in the brain and of CXCL10 in the cerebrospinal fluid. The latter parameter and sleep/wake alterations could provide combined humoral and functional biomarkers of the early encephalitic stage in African trypanosomiasis

The aging brain, neuroinflammatory signaling and sleep-wake regulation

Tissues and organs change over time, regulated by intrinsic (genetic) determinants and environmental (and microenvironmental) adaptation. Brain changes during lifetime are especially critical, as the brain is the effector of cognition and the vast majority of neurons live throughout the life of the individual. In addition, brain aging mechanisms are especially critical for disease vulnerability, given the aging­related prevalence of pathologies that include neurodegenerative diseases. In this context, the present contribution concisely highlights data yielded by recent trends of research on the normal aging brain, and specifically: the occurrence of synaptic changes (rather than neuronal loss) and the altered regulation of adult neurogenesis (which represents a novel exciting field of knowledge); the development of a low­grade chronic inflammatory state which primes glial cells and may lead to changes in intercellular crosstalk, thus playing a potential role in the brain susceptibility to neurodegeneration; changes occurring in state­dependent behavior, sleep and wake, which are products of global brain functioning and underlie consciousness and cognitive performance; changes in the biological clock, the hypothalamic suprachiasmatic nucleus, which regulates sleep­wake alternation and other endogenous rhythms. Altogether, the present synopsis of recent studies at the molecular, cellular, and functional levels emphasizes the idea that the normal aging brain should be viewed as an example of adaptation and plasticity rather than as an obligatory decline

Emerging data supporting stromal cell therapeutic potential in cancer: reprogramming stromal cells of the tumor microenvironment for anti-cancer effects

After more than a decade of controversy on the role of stromal cells in the tumor microenvironment, the emerging data shed light on pro-tumorigenic and potential anti-cancer factors, as well as on the roots of the discrepancies. We discuss the pro-tumorigenic effects of stromal cells, considering the effects of tumor drivers like hypoxia and tumor stiffness on these cells, as well as stromal cell-mediated adiposity and immunosuppression in the tumor microenvironment, and cancer initiating cells' cellular senescence and adaptive metabolism. We summarize the emerging data supporting stromal cell therapeutic potential in cancer, discuss the possibility to reprogram stromal cells of the tumor microenvironment for anti-cancer effects, and explore some causes of discrepancies on the roles of stromal cells in cancer in the available literature

Tumor Microenvironment Uses a Reversible Reprogramming of Mesenchymal Stromal Cells to Mediate Pro-tumorigenic Effects

The role of mesenchymal stromal cells (MSCs) in the tumor microenvironment is well described. Available data support that MSCs display anticancer activities, and that their reprogramming by cancer cells in the tumor microenvironment induces their switch toward pro-tumorigenic activities. Here we discuss the recent evidence of pro-tumorigenic effects of stromal cells, in particular (i) MSC support to cancer cells through the metabolic reprogramming necessary to maintain their malignant behavior and stemness, and (ii) MSC role in cancer cell immunosenescence and in the establishment and maintenance of immunosuppression in the tumor microenvironment. We also discuss the mechanisms of tumor microenvironment mediated reprogramming of MSCs, including the effects of hypoxia, tumor stiffness, cancer-promoting cells, and tumor extracellular matrix. Finally, we summarize the emerging strategies for reprogramming tumor MSCs to reactivate anticancer functions of these stromal cells

Developmental pathways associated with cancer metastasis: Notch, Wnt, and Hedgehog

Master developmental pathways, such as Notch, Wnt, and Hedgehog, are signaling systems that control proliferation, cell death, motility, migration, and stemness. These systems are not only commonly activated in many solid tumors, where they drive or contribute to cancer initiation, but also in primary and metastatic tumor development. The reactivation of developmental pathways in cancer stroma favors the development of cancer stem cells and allows their maintenance, indicating these signaling pathways as particularly attractive targets for efficient anticancer therapies, especially in advanced primary tumors and metastatic cancers. Metastasis is the worst feature of cancer development. This feature results from a cascade of events emerging from the hijacking of epithelial-mesenchymal transition, angiogenesis, migration, and invasion by transforming cells and is associated with poor survival, drug resistance, and tumor relapse. In the present review, we summarize and discuss experimental data suggesting pivotal roles for developmental pathways in cancer development and metastasis, considering the therapeutic potential. Emerging targeted antimetastatic therapies based on Notch, Wnt, and Hedgehog pathways are also discussed

Actigraphy in Human African Trypanosomiasis as a Tool for Objective Clinical Evaluation and Monitoring: A Pilot Study

The clinical picture of the parasitic disease human African trypanosomiasis (HAT, also called sleeping sickness) is dominated by sleep alterations. We here used actigraphy to evaluate patients affected by the Gambiense form of HAT. Actigraphy is based on the use of battery-run, wrist-worn devices similar to watches, widely used in middle-high income countries for ambulatory monitoring of sleep disturbances. This pilot study was motivated by the fact that the use of polysomnography, which is the gold standard technology for the evaluation of sleep disorders and has greatly contributed to the objective identification of signs of disease in HAT, faces tangible challenges in resource-limited countries where the disease is endemic. We here show that actigraphy provides objective data on the severity of sleep-wake disturbances that characterize HAT. This technique, which does not disturb the patient's routine activities and can be applied at home, could therefore represent an interesting, non-invasive tool for objective HAT clinical assessment and long-term monitoring under field conditions. The use of this method could provide an adjunct marker of HAT severity and for treatment follow-up, or be evaluated in combination with other disease biomarkers in body fluids that are currently under investigation in many laboratories

SLEEP AND WAKE ALTERATIONS IN AFRICAN TRYPANOSOMIASIS: FUNCTIONAL AND HISTOPATHOLOGICAL STUDY

La tripanosomiasi umana africana (human African trypanosomiasis o HAT), denominata anche malattia del sonno, è una grave malattia infiammatoria causata dal parassita Trypanosoma brucei (T.b.). Questa patologia, che è una tipica malattia “negletta”, è caratterizzata da alterazioni della struttura del sonno e dell’alternanza sonno-veglia. Il primo stadio, emolinfatico, della malattia, evolve nel secondo stadio, meningoencefalitico, quando il parassita T.b. attraversa la barriera emato-encefalica (BEE) ed invade il paraenchima cerebrale. I meccanismi patogenetici delle alterazioni neurologiche causate dall’infezione, così come la diagnosi di stadio quando il paziente si presenta all’osservazione e l’evoluzione della malattia rivestono alte valenze traslazionali poiché hanno importanti implicazioni diagnostiche e terapeutiche. Gli studi sulla tripanosomiasi africana che vengono presentati in questa tesi si articolano in 4 parti. Il primo studio sperimentale si è focalizzato, in ratti e topi infettati sperimentalmente con T.b. brucei, sull’orexina, un neuropeptide che svolge un ruolo chiave nel mantenimento dello stato di veglia e nelle transizioni sonno-veglia, e su un altro peptide, il melanin-concentrating hormone (MCH), implicato nella regolazione del sonno. Lo studio immunocitochimico ha rivelato una diminuzione, in una fase avanzata dell’infezione, sia delle cellule ipotalamiche che esprimono orexina che di quelle che esprimono MCH. L’indagine del livello di orexina nel liquor dei ratti infetti non ha, tuttavia, documentato alterazioni significative di tale parametro nel corso dell’infezione, indicando quindi che la concentrazione di orexina nel liquor non è un parametro atto a fornire un biomarcatore della malattia. Di particolare interesse patogenetico è il dato, rivelato da questo stesso studio, di un’alterata regolazione dell’attività dei neuroni orexinergici (valutata mediante l’oscillazione spontanea, nelle 24h, dell’espressione di Fos in tali cellule) durante il giorno e la notte. Il progetto si è poi rivolto alla ricerca, nell’infezione sperimentale nel ratto, di alterazioni funzionali che possano riflettere (e quindi consentire di svelare) il passaggio del T.b. attraverso la BEE. L’evoluzione temporale dell’infiltrazione di parassiti e linfociti nel parenchima encefalico è stata indagata mediante marcature immunoistochimiche multiple. Questa sperimentazione ha rivelato che l’ingresso del parassita e delle cellule T si verifica prevalentemente attraverso l’ipotalamo posteriore. Tale dato suggerisce differenze regionali di permeabilità della BEE dovute a segnali infiammatori nel corso dell’infezione, così come una potenziale vulnerabilità a questi eventi di gruppi neuronali implicati nella regolazione del sonno e della veglia. Gli stati di sonno e veglia ed i cicli della temperatura corporea e attività-riposo nelle 24h sono stati quindi monitorati di continuo, in ratti infetti, mediante registrazione telemetrica e paragonati con gli stessi parametri in condizioni basali prima dell’infezione. Analisi dettagliate di questa messe di dati hanno rivelato che alterazioni della struttura del sonno iniziano precocemente dopo l’infezione e precedono la neuroinvasione del parassita. Tali dati hanno svelato, inoltre, i parametri temporali dell’insorgenza e della progressione di alterazioni del sonno e della veglia, così come dei ritmi della temperatura corporea e attività-riposo, e le loro caratteristiche nel corso dell’infezione. Le analisi degli ipnogrammi ed actogrammi degli animali infetti hanno dimostrato che le alterazioni del ritmo attività-riposo possono fornire misurazioni attendibili delle alterazioni di sonno e veglia nel corso dell’infezione. Questo studio ha aperto la strada all’ultima parte del progetto, attuata in collaborazione con l’Università di Yaoundé 1 (Cameroon), nella quale sono state analizzate registrazioni actigrafiche e polisonnografiche di pazienti affetti da HAT. E’ stato così determinato che l’actigrafia può fornire, in questa patologia, un utile strumento di indagine atto a monitorare la gravità della malattia, aprendo così nuove prospettive per la valutazione clinica dei pazienti.Human African trypanosomiasis (HAT), also called sleeping sickness, is a neglected, severe neuroinflammatory disease caused by the protozoan Trypanosoma brucei (T.b.), and is characterized by alterations of the sleep pattern and sleep-wake cycle. The first, hemolymphatic stage of the disease evolves into the second, meningoencephalitic stage when T.b. cross the blood-brain barrier (BBB) and invade the brain parenchyma. Pathogenetic mechanisms of brain dysfunction caused by the infection, as well as knowledge of disease stage timing and evolution, are of high relevance also from the translational point of view given their diagnostic and therapeutic implications. The present doctoral studies on African trypanosomiasis have been articulated in 4 steps. The first investigation focused on orexin, a neuropeptide which plays a key role in wakefulness and in stabilizing sleep-wake transitions, as well as melanin concentrating hormone (MCH), which plays a role in sleep regulation, in rats and mice infected with T.b. brucei. Immunocytochemical study of orexin and MCH revealed a decrease, at an advanced stage of infection, of hypothalamic cells expressing either peptides; orexin level in the cerebrospinal fluid was not, however, significantly affected, and thus cannot provide a disease biomarker. Interestingly, day/night activity (as shown by Fos expression) of orexin-containing neurons was found to be deregulated in infected animals. Further steps of the project focused on search for functional changes which could reflect T.b. passage across the BBB, and these were investigated in the above rat model. The timing of parasite neuroinvasion and T-cell recruitment in the brain parenchyma was determined in multiple labeling immunocytochemical investigations. This part of the study revealed a prevalence of both parasite and T-cell entry through the posterior hypothalamus, suggesting regional differences in BBB permeability due to inflammatory signaling during the infection, and a potential vulnerability of hypothalamic sleep-wake regulatory cell groups to these events. Sleep-wake stages, core body temperature and rest-activity during 24 h were then continuously monitored in infected rats with telemetric recording and compared to baseline data. Extensive analyses of such data revealed that sleep structure alterations start early after the infection and precede parasite neuroinvasion, and showed then the onset and progression of distinct changes of wake and sleep states, as well as of body temperature and rest-activity rhythms. Furthermore, analyses of the hypnograms and actograms of the infected animals showed that rest-activity changes can provide reliable measurements of sleep-wake alterations. This paved the way to the last part of the project, pursued in collaboration with the University of Yaoundé 1 (Cameroon), in which hypnograms and actigraphic recordings of HAT patients have been analyzed. It has thus been determined that actigraphy can provides a useful tool for disease severity monitoring, opening novel perspectives for the clinical evaluation of patients with a non-invasive technique

Effect of inflammatory challenge on hypothalamic neurons expressing orexinergic and melanin-concentrating hormone

Neurons containing the hypothalamic peptides orexin-A (hypocretin 1) and melanin-concentrating hormone (MCH) have been reported numerous roles in the regulation of the sleep-wake cycle, energy balance and feeding behavior. We investigated the response of these cells to repeated administration of low doses of endotoxin lipopolysaccharide (LPS) in mice. Adult male C57/6J mice where intraperitoneally (i.p.) injected with either LPS or phosphate-buffered saline (PBS) weekly for either 4 or 8 weeks, and afterwards were sacrificed at different time intervals from last injection. A significant drop in orexin-containing neuron number, but not in numbers of MCH or neuronal nuclear antigen (NeuN)-immunoreactive neurons, was observed after 8 weeks of LPS treatment, as compared to PBS treatment. Orexin expression entirely returned to control levels 30 days after the last LPS injection in mice treated for 8 weeks. These data strongly suggest the occurrence of selective alterations of orexinergic system, reversible over time, following repeated and intermittent systemic inflammatory challenge in mice