Laser ablation-inductively coupled plasma mass spectrometry: an emerging technology for detecting rare cells in tissue sections.

Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 10(9) particles/ml for 1 h incorporated an average of 3.39 × 10(8) Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 10(5) counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific (154)Sm-tagged anti-HLA-DR or (174)Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology.

Langerhans cells promote early germinal center formation in response to Leishmania-derived cutaneous antigens.

Efficient formation of early GCs depends on the close interaction between GC B cells and antigen-primed CD4(+) follicular helper T cells (TFH ). A tight and stable formation of TFH /B cell conjugates is required for cytokine-driven immunoglobulin class switching and somatic hypermutation of GC B cells. Recently, it has been shown that the formation of TFH /B cell conjugates is crucial for B-cell differentiation and class switch following infection with Leishmania major parasites. However, the subtype of DCs responsible for TFH -cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin(+) DC subsets in vivo, we show that the functionality of TFH /B cell conjugates is disturbed after depletion of Langerhans cells (LCs): LC-depleted mice show a reduction in somatic hypermutation in B cells isolated from TFH /B cell conjugates and markedly reduced GC reactions within skin-draining lymph nodes. In conclusion, this study reveals an indispensable role for LCs in promoting GC B-cell differentiation following cutaneous infection with Leishmania major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.

Leishmania major infection in humanized mice induces systemic infection and provokes a nonprotective human immune response.

BACKGROUND: Leishmania (L.) species are the causative agent of leishmaniasis. Due to the lack of efficient vaccine candidates, drug therapies are the only option to deal with cutaneous leishmaniasis. Unfortunately, chemotherapeutic interventions show high toxicity in addition to an increased risk of dissemination of drug-resistant parasites. An appropriate laboratory animal based model is still missing which allows testing of new drug strategies in the context of human immune cells in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Humanized mice were infected subcutaneously with stationary phase promastigote L. major into the footpad. The human immune response against the pathogen and the parasite host interactions were analyzed. In addition we proved the versatility of this new model to conduct drug research studies by the inclusion of orally given Miltefosine. We show that inflammatory human macrophages get infected with Leishmania parasites at the site of infection. Furthermore, a Leishmania-specific human-derived T cell response is initiated. However, the human immune system is not able to prevent systemic infection. Thus, we treated the mice with Miltefosine to reduce the parasitic load. Notably, this chemotherapy resulted in a reduction of the parasite load in distinct organs. Comparable to some Miltefosine treated patients, humanized mice developed severe side effects, which are not detectable in the classical murine model of experimental leishmaniasis. CONCLUSIONS/SIGNIFICANCE: This study describes for the first time L. major infection in humanized mice, characterizes the disease development, the induction of human adaptive and innate immune response including cytokine production and the efficiency of Miltefosine treatment in these animals. In summary, humanized mice might be beneficial for future preclinical chemotherapeutic studies in systemic (visceral) leishmaniasis allowing the investigation of human immune response, side effects of the drug due to cytokine production of activated humane immune cells and the efficiency of the treatment to eliminate also not replicating ("hiding") parasites.

Leishmania major parasite stage-dependent host cell invasion and immune evasion.

Leishmania pathogenesis is primarily studied using the disease-inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease-relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage-specific DsRed protein expression. We found parasite stage-specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote-specific complement receptor 3-mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1-mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage-specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease-propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigote-driven immune response in leishmaniasis.

An advanced approach for the characterization of dendritic cell-induced T cell proliferation in situ.

It is commonly accepted that dendritic cells (DCs) play a pivotal role in the induction of adaptive T cell-mediated immune responses. The clonal expansion of antigen-specific T cells within secondary lymphoid organs reflects the efficiency of antigen processing and presentation by DCs. Consequently, the quantification of proliferating antigen-specific T cells represents an important read-out for analyzing DC-T cell interactions. Standard proliferation assays are usually performed with cell suspensions of antigen-stimulated lymphocytes labelled with carboxyfluorescein diacetate succinimidyl ester, [(3)H] thymidine, or 5-bromo-2-deoxyuridine. However, these assays have important limitations, including the complete loss of information about the localization of proliferating cells in situ. Additionally, the ex vivo stimulation with antigen does not necessarily reflect the in vivo situation of cell proliferation. Therefore, an advanced approach for a detailed characterization of proliferating T cells in situ would be helpful for the interpretation of ongoing adaptive immune responses. Here, we describe the development of a fluorescence-based multicolour assay allowing the characterization and quantification of proliferation in different T cell subtypes in cryosections of lymphoid organs. Our approach combines the major benefits of flow cytometry and immunofluorescent-based histology, such as (i) multicolour phenotyping of cell populations and (ii) description of tissue-specific sites of cell proliferation.

Identification of neural crest-derived stem cell-like cells from the corneal limbus of juvenile mice.

The neural crest is a transient embryonic tissue alongside the lateral margins of the neural folds. It contains cells involved in the development of anterior eye segments such as the cornea. Previous studies have revealed the presence of neural crest-derived stem cells in the cornea of the adult murine eye. However, less is known about cell populations of the developing eye. In this study, we have identified neural crest-derived murine corneal cells (MCCs) with stem cell-like properties derived from the corneal limbus of mice between postnatal days 1 and 8. RT-PCR analysis and immunofluorescence staining demonstrate that MCCs express a unique profile of markers including typical neural crest-originated stem cell transcripts like Sca1. MCCs show a limited self-renewing capacity but possess multipotency under in vitro conditions after differentiation into cells with features resembling adipocytes, osteoblasts and neuronal cells. Interestingly, MCCs could not be isolated from corneas of adult animals. We conclude that MCCs are stem cell-like cells of an early postnatal period of murine eye growth, probably involved in the early development of the postnatal cornea.

Gene expression of nestin, collagen type I and type III in human dental follicle cells after cultivation in serum-free medium.

BACKGROUND: Undifferentiated human dental cells and especially human dental follicle cells are interesting for potential dental treatments. These somatic stem cells are cultured usually in cell culture medium containing bovine serum. In the age of bovine spongiform encephalopathy (BSE), a serum-free cell culture system for dental follicle cells are recommended, if these cells will be applied in dentistry. PURPOSE: However, less is known about the cultivation of dental follicle cells in serum-replacement medium. In this study, we cultivated dental follicle cells in serum-free cell culture medium, which is normally applied for neuronal stem/progenitor cells. MATERIALS AND METHODS: Dental follicle cells were cultivated in both serum-free and serum-containing cell culture media, and gene expression profiles were recorded for connective tissue markers collagen type I and type III and for the human dental follicle cell marker nestin. RESULTS: It is interesting to note that the gene expressions of collagens and nestin were similar after applying both cell culture conditions. CONCLUSION: Although the gene expression of dental follicle cell markers was unchanged, a more appropriate serum-free cell culture medium is recommended for cell proliferation of dental follicle cells.

Murine Müller cells are progenitor cells for neuronal cells and fibrous tissue cells.

Mammalian Müller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Müller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Müller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Müller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Müller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.

Evaluación in vitro de la resistencia a ciprofloxacina en biopelículas y poblaciones planctónicas de pseudomonas aeruginosa de origen hospitalario / In vitro determination of ciprofloxacin resistance in Pseudomonas aeruginosa and hospital planktonic populations

Objetivo: Evaluar la susceptibilidad / resistencia in vitro de biopelículas y poblaciones planctónicas de Pseudomonas aeruginosa de origen hospitalario a la ciprofloxacina y compararlas con los resultados con las pruebas de susceptibilidad estándar obtenidos. Diseño: Estudio microbiológico. Lugar: Laboratorio de Microbiología Molecular, Facultad de Ciencias Biológicas, UNMSM. Material biológico: Cepas de Pseudomonas aeruginosa. Intervenciones: En cepas resistentes y sensibles obtenidas del Hospital del Niño, se realizó pruebas de concentración mínima inhibitoria (MIC); luego, se procedió a formar las biopelículas, en membranas de filtración y cultivos planctónicos en caldo, para ser sometidos ambos a diferentes concentraciones de ciprofloxacina y realizar el conteo en placas a intervalos de 40 minutos y a las 24 horas de exposición. Principales medidas de resultados: Susceptibilidad / resistencia in vitro de biopelículas y poblaciones planctónicas de Pseudomonas aeruginosa de origen hospitalario. Resultados: Al examinar la cepa sensible, ésta incrementa suresistencia en más de 200 veces, bajo la forma de biopelícula, encontrándose una diferencia poco significativa en las cepas resistentes, ya que la población comienza a disminuir alrededor de una concentración de 128 μg/mL de la droga. Conclusiones: Una misma cepa, como en el caso de la Pseudomona aeruginosa, tiene un comportamiento distinto a la ciprofloxacina, de acuerdo a su forma de crecimiento. Se recomienda la determinación del MIC en biopelículas de agentes etiológicos de infecciones recurrentes y crónicas que desarrollan el estado de biopelícula.

Objective: To assess in vitro sensitivity / resistance of biofilms and planktonic populations of Pseudomonas aeruginosa from a hospital environment to ciprofloxacin and to compare them against the results of standard susceptibility test obtained. Design: Microbiological study. Setting: Molecular Microbiology Laboratory, Faculty of Biological Sciences, UNMSM. Biologic material: Pseudomonas aeruginosa strains. Interventions: Resistant and susceptible strains obtained form Hospital del Niño were tested for minimum inhibitory concentration (MIC); then biofilms were formed on filtration membranes, and planktonic cultures in broth were tested with different ciprofloxacin concentrations, with plate count at 40 minutes intervals and 24 hours after exposure. Main outcome measures: In vitro susceptibility / resistance of hospital origin Pseudomonas aeruginosa biofilms and planktonic populations. Results: Sensitive strains increase their resistance more than 200 times under biofilm growth; no significant difference was found with resistant strains and both start to decrease their population at about 128 ìg/Ml drug concentration. Conclusions: The same strain, as in case of Pseudomona aeruginosa, has a different response to ciprofloxacin, according to its growth. A MIC biofilm test of recurrent and chronic infections etiological agents is recommended.