Specific Cell Targeting Therapy Bypasses Drug Resistance Mechanisms in African Trypanosomiasis

African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.JAGS was funded by the European Union, grant FP7-HEALTH-2007-B-2.3.4-1.223048, NANOTRYP and Ministerio de Economía y Competitividad, Spain Plan Nacional de Investigación grant SAF2011- 30528. JLA was funded by Instituto de Salud Carlos III, Spain, grant FIS. 11/02571. HPdK was supported by a grant from the Medical Research Council (84733)

Attenuation of the Sensing Capabilities of PhoQ in Transition to Obligate Insect–Bacterial Association

Sodalis glossinidius, a maternally inherited endosymbiont of the tsetse fly, maintains genes encoding homologues of the PhoP-PhoQ two-component regulatory system. This two-component system has been extensively studied in facultative bacterial pathogens and is known to serve as an environmental magnesium sensor and a regulator of key virulence determinants. In the current study, we show that the inactivation of the response regulator, phoP, renders S. glossinidius sensitive to insect derived cationic antimicrobial peptides (AMPs). The resulting mutant strain displays reduced expression of genes involved in the structural modification of lipid A that facilitates resistance to AMPs. In addition, the inactivation of phoP alters the expression of type-III secretion system (TTSS) genes encoded within three distinct chromosomal regions, indicating that PhoP-PhoQ also serves as a master regulator of TTSS gene expression. In the absence of phoP, S. glossinidius is unable to superinfect either its natural tsetse fly host or a closely related hippoboscid louse fly. Furthermore, we show that the S. glossinidius PhoQ sensor kinase has undergone functional adaptations that result in a substantially diminished ability to sense ancestral signals. The loss of PhoQ's sensory capability is predicted to represent a novel adaptation to the static symbiotic lifestyle, allowing S. glossinidius to constitutively express genes that facilitate resistance to host derived AMPs

Immunological determinants in a mouse model of chemical-induced asthma after multiple exposures

In a mouse model of chemical-induced asthma, we investigated the effects of multiple challenges, using toluene diisocyanate (TDI), a known cause of occupational asthma. On days 1 and 7, BALB/c mice received TDI or vehicle (acetone/olive oil). On days 10, 13 and 16 the mice received an intranasal instillation of TDI. Ventilatory function (Penh) was monitored by whole body plethysmography for 40 min after each challenge. Reactivity to methacholine was measured 22 h later. Pulmonary inflammation, TNF-\u3b1 and MIP-2 levels were assessed 24 h after the last challenge by broncho-alveolar lavage (BAL). Other immunological parameters included total IgE, lymphocyte sub-populations in auricular and cervical lymph nodes, and IL-4, IFN-\u3b3 and IL-13 levels in supernatants of lymph node cells, cultured with or without concanavalin A. Early ventilatory function and airway reactivity increased in all groups that received a dermal application and one or multiple intranasal challenges of TDI. After multiple challenges, lung inflammation was characterized by neutrophils ( 3c15%), and eosinophils ( 3c4%), along with an increase in BAL MIP-2 and TNF-\u3b1 levels. The auricular and cervical lymph node cells of all sensitized mice showed an increase in B cells, Th cells and an increased concentration of in vitro release of IL-4, IFN-\u3b3 and IL-13 after stimulation with concanavalin A. Total serum IgE was elevated in dermally TDI-sensitized mice. This protocol including multiple challenges results in a model that resembles human asthma, indicating that responses found in the model using a single challenge could be a good first indication for the development of asthma

Validation of a mouse model of chemical-induced asthma using trimellitic anhydride, a respiratory sensitizer, and dinitrochlorobenzene, a dermal sensitizer

Background: Occupational asthma can be caused by chemicals. Previously, we established a murine model of immunologically mediated chemical-induced asthma using toluene diisocyanate. Objective: We sought to verify this model using trimellitic anhydride (TMA), a respiratory sensitizer, and 1-chloro-2,4-dinitrobenzene (DNCB), a dermal sensitizer. Methods: BALB/c mice received dermal applications (vehicle or chemical) on days 1 and 7. On day 10, they received an intranasal instillation (vehicle or chemical). Whole-body plethysmography (enhanced pause) was used to monitor changes in ventilatory function and methacholine reactivity. Pulmonary inflammation was assessed by using bronchoalveolar lavage (cells, TNF-\u3b1 levels, and macrophage inflammatory protein 2 levels). Immunologic parameters included total serum IgE levels, lymphocyte distribution in auricular and cervical lymph nodes, and IL-4 and IFN-\u3b3 levels in supernatants of lymph node cells incubated with or without concanavalin A. Results: Mice dermally treated and intranasally challenged with TMA experienced markedly increased enhanced pause immediately after intranasal challenge and increased methacholine reactivity (24 hours later). Mice similarly treated with DNCB did not show any ventilatory changes. Neutrophil influx and increased macrophage inflammatory protein 2 and TNF-\u3b1 levels were found in bronchoalveolar lavage fluid in both TMA- and DNCB-treated mice. The proportion of CD19 + B cells was increased in auricular and cervical lymph nodes of TMA-treated mice. IL-4 and IFN-\u3b3 levels were increased in supernatants of concanavalin A-stimulated auricular and cervical lymph node cells of TMA- or DNCB-treated mice; however, the relative proportions of IL-4 and IFN-\u3b3 levels differed between TMA- and DNCB-treated mice. Serum total IgE levels were increased in TMA-treated mice only. Conclusion: Both compounds induce a mixed T H1-T H2 response, but only TMA induced ventilatory changes. Clinical implications: In the workplace avoiding skin contact with chemical sensitizers might be advised to prevent chemical-induced asthma

Immunological determinants of ventilatory changes induced in mice by dermal sensitization and respiratory challenge with toluene diisocyanate

The objective of the study was to characterize better the immunologic mechanisms underlying a previously developed animal model of chemical-induced asthma. BALB/c and severe combined immunodeficiency disease (SCID) mice received toluene diisocyanate (TDI) or vehicle on each ear on day 1 and/or day 7. On day 10, they were intranasally challenged with TDI or vehicle. Ventilatory function was monitored by whole body plethysmography for 40 min after challenge. Reactivity to methacholine was measured 23 h later: enhanced pause and actual resistance measurements. Pulmonary inflammation was assessed 1, 6, and 24 h after challenge by bronchoalveolar lavage (BAL). Tumor necrosis factor-\u3b1 and macrophage inflammatory protein (MIP)-2 levels were measured in BAL. Immunological parameters included total IgE, IgG1, and IgG2a in serum, lymphocyte populations in auricular and cervical lymph nodes, and IL-4 and IFN-\u3b3 levels in supernatants of lymph node cells, cultured with or without concanavalin A. Ventilatory changes suggestive of airway obstruction and increased methacholine reactivity were observed in all TDI-sensitized and TDI intranasally instilled mice, except in SCID mice. A neutrophil influx, accompanied by an increase in MIP-2 levels, was found in BAL of all responding groups 6 and 24 h after intranasal challenge. In BALB/c mice an increased level of CD19+ B cells was found in the auricular lymph nodes. IL-4 and IFN-\u3b3 levels were increased in supernatants of concanavalin A-stimulated auricular lymph node cells from BALB/c mice completely treated with TDI. These results indicate that our model is dependent on the presence of lymphocytes, but it is not characterized by a preferential stimulation of Th1 or Th2 lymphocytes. Copyright \ua9 2007 the American Physiological Society

What's lab : Bepalingsinformatie anno 2014

In het voorjaar van 2013 is bij het Laboratorium Klinische Chemie en Haematologie (LKCH) van het UMC Utrecht een begin gemaakt met het project ‘Centralisatie bepalingsgegevens’. Dit project werd ingegeven door het feit dat allerlei bepalingsgegevens (uiteenlopend van bepalingsfrequentie en kostprijs, tot analysemethode en -duur, referentiewaarden, voorkeursmateriaal en contactpersonen) op dat moment op tal van verschillende plekken opgezocht moest worden (SOPs, de bepalingencatalogus van de afdeling op het intranet, GLIMS, etc.) waarbij niet alleen de vindbaarheid maar ook de actualiteit zowel voor eigen medewerkers als artsen en verpleegkundigen regelmatig te wensen overliet. Daarnaast was de wijze waarop de informatie gepresenteerd werd niet meer van deze tijd. In een half jaar tijd is een nieuwe website gebouwd waarbij informatie uit het laboratoriuminformatiesysteem (LIS) op een overzichtelijke wijze toegankelijk is gemaakt voor alle gebruikers. Deze website is sinds december 2013 in de lucht en bij zijn introductie ‘What’s Lab’ gedoopt (www.whatslab.nl)

What's lab : Bepalingsinformatie anno 2014

In het voorjaar van 2013 is bij het Laboratorium Klinische Chemie en Haematologie (LKCH) van het UMC Utrecht een begin gemaakt met het project ‘Centralisatie bepalingsgegevens’. Dit project werd ingegeven door het feit dat allerlei bepalingsgegevens (uiteenlopend van bepalingsfrequentie en kostprijs, tot analysemethode en -duur, referentiewaarden, voorkeursmateriaal en contactpersonen) op dat moment op tal van verschillende plekken opgezocht moest worden (SOPs, de bepalingencatalogus van de afdeling op het intranet, GLIMS, etc.) waarbij niet alleen de vindbaarheid maar ook de actualiteit zowel voor eigen medewerkers als artsen en verpleegkundigen regelmatig te wensen overliet. Daarnaast was de wijze waarop de informatie gepresenteerd werd niet meer van deze tijd. In een half jaar tijd is een nieuwe website gebouwd waarbij informatie uit het laboratoriuminformatiesysteem (LIS) op een overzichtelijke wijze toegankelijk is gemaakt voor alle gebruikers. Deze website is sinds december 2013 in de lucht en bij zijn introductie ‘What’s Lab’ gedoopt (www.whatslab.nl)