The CXCR3-CXCL11 signaling axis mediates macrophage recruitment and dissemination of mycobacterial infection

The recruitment of leukocytes to infectious foci depends strongly on the local release of chemoattractant mediators. The human CXC chemokine receptor 3 (CXCR3) is an important node in the chemokine signaling network and is expressed by multiple leukocyte lineages, including T cells and macrophages. The ligands of this receptor originate from an ancestral CXCL11 gene in early vertebrates. Here, we used the optically accessible zebrafish embryo model to explore the function of the CXCR3-CXCL11 axis in macrophage recruitment and show that disruption of this axis increases the resistance to mycobacterial infection. In a mutant of the zebrafish ortholog of CXCR3 (cxcr3.2), macrophage chemotaxis to bacterial infections was attenuated, although migration to infection-independent stimuli was unaffected. Additionally, attenuation of macrophage recruitment to infection could be mimicked by treatment with NBI74330, a high-affinity antagonist of CXCR3. We identified two infection-inducible CXCL11-like chemokines as the functional ligands of Cxcr3.2, showing that the recombinant proteins exerted a Cxcr3.2-dependent chemoattraction when locally administrated in vivo. During infection of zebrafish embryos with Mycobacterium marinum, a well-established model for tuberculosis, we found that Cxcr3.2 deficiency limited the macrophage-mediated dissemination of mycobacteria. Furthermore, the loss of Cxcr3.2 function attenuated the formation of granulomatous lesions, the typical histopathological features of tuberculosis, and led to a reduction in the total bacterial burden. Prevention of mycobacterial dissemination by targeting the CXCR3 pathway, therefore, might represent a host-directed therapeutic strategy for treatment of tuberculosis. The demonstration of a conserved CXCR3-CXCL11 signaling axis in zebrafish extends the translational applicability of this model for studying diseases involving the innate immune system

Optimizing Staining Protocols for Laser Microdissection of Specific Cell Types from the Testis Including Carcinoma In Situ

Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdissection technology allows for enrichment of specific cell types. However, when the cells are not morphologically distinguishable, it is necessary to use a specific staining method for the target cells. In this study we have tested different fixatives, storage conditions for frozen sections and staining protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma in situ cells, and other cells with embryonic stem cell-like properties that express the alkaline phosphatase, and one for specific staining of lipid droplet-containing cells, which is useful for isolation of the androgen-producing Leydig cells. Both protocols retain a morphology that is compatible with laser microdissection and yield RNA of a quality suitable for PCR and microarray analysis

High Resolution Spectral Domain Optical Coherence Tomography (SD-OCT) in Multiple Sclerosis: The First Follow Up Study over Two Years

“Non-invasive, faster and less expensive than MRI” and “the eye is a window to the brain” are recent slogans promoting optical coherence tomography (OCT) as a new surrogate marker in multiple sclerosis (MS). Indeed, OCT allows for the first time a non-invasive visualization of axons of the central nervous system (CNS). Reduction of retina nerve fibre layer (RNFL) thickness was suggested to correlate with disease activity and duration. However, several issues are unclear: Do a few million axons, which build up both optic nerves, really resemble billions of CNS neurons? Does global CNS damage really result in global RNFL reduction? And if so, does global RNFL reduction really exist in all MS patients, and follow a slowly but steadily ongoing pattern? How can these (hypothesized) subtle global RNFL changes be reliably measured and separated from the rather gross RNFL changes caused by optic neuritis? Before generally being accepted, this interpretation needs further critical and objective validation.We prospectively studied 37 MS patients with relapsing remitting (n = 27) and secondary progressive (n = 10) course on two occasions with a median interval of 22.4±0.5 months [range 19–27]. We used the high resolution spectral domain (SD-)OCT with the Spectralis 3.5 mm circle scan protocol with locked reference images and eye tracking mode. Patients with an attack of optic neuritis within 12 months prior to the onset of the study were excluded.Although the disease was highly active over the observation period in more than half of the included relapsing remitting MS patients (19 patients/32 relapses) and the initial RNFL pattern showed a broad range, from normal to markedly reduced thickness, no significant changes between baseline and follow-up examinations could be detected.These results show that caution is required when using OCT for monitoring disease activity and global axonal injury in MS

Immunocytochemically detected free peritoneal tumour cells (FPTC) are a strong prognostic factor in gastric carcinoma

We prospectively investigated the prognostic significance of free peritoneal tumour cells (FPTC) in a series of 118 patients with completely resected gastric carcinoma. Immunocytochemistry with the monoclonal antibody Ber-Ep4 was performed on cytospins from intraoperative peritoneal lavage specimens. Twenty-three patients (20%) had FPTC which was significantly correlated with pT and pN categories, stage, tumour size, lymphatic invasion, Laurèn and WHO classifications and perigastric adipose tissue metastases. The median survival time for all FPTC positive compared with negative patients was significantly shorter (11 compared with > 72 months), with estimated 5-year survival rates of 8% vs. 60%. None of the patients with FPTC had an early gastric cancer. In advanced tumour subgroups without and with serosal invasion (n = 59 and 35), there were 19% and 34% with FPTC. Multivariate survival analysis showed nodal status, FPTC, mesenteric lymphangiosis, and lymph node metastasis to the compartment III to be independent prognostic factors with relative risks of 6.6, 4.5, 2.9 and 2.2 respectively. Recurrent disease occurred in 91% of FPTC-positive and in 38% of FPTC-negative patients. FPTC had a positive predictive value of 91% and a specificity of 97% for tumour recurrence. FPTC is a strong negative, independent prognostic indicator for survival in gastric carcinoma. © 1999 Cancer Research Campaig

In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

BACKGROUND: Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates

Fragile x syndrome and autism: from disease model to therapeutic targets

Autism is an umbrella diagnosis with several different etiologies. Fragile X syndrome (FXS), one of the first identified and leading causes of autism, has been modeled in mice using molecular genetic manipulation. These Fmr1 knockout mice have recently been used to identify a new putative therapeutic target, the metabotropic glutamate receptor 5 (mGluR5), for the treatment of FXS. Moreover, mGluR5 signaling cascades interact with a number of synaptic proteins, many of which have been implicated in autism, raising the possibility that therapeutic targets identified for FXS may have efficacy in treating multiple other causes of autism

Regulatory T cells and their role in rheumatic diseases: a potential target for novel therapeutic development

Regulatory T cells have an important role in limiting immune reactions and are essential regulators of self-tolerance. Among them, CD4+CD25high regulatory T cells are the best-described subset. In this article, we summarize current knowledge on the phenotype, function, and development of CD4+CD25high regulatory T cells. We also review the literature on the role of these T cells in rheumatic diseases and discuss the potential for their use in immunotherapy

Different requirements for plaminogen activator inhibitor 1 (PAI-1) loop insertion during latency transition and target proteinase inhibition as evidenced by phage-displayed hypermutated PAI-1 libraries

Plasminogen activator inhibitor type 1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily. Its highly mobile reactive-center loop (RCL) is thought to account for both the rapid inhibition of tissue-type plasminogen activator (t-PA), and the rapid and spontaneous transition of the unstable, active form of PAI-1 into a stable, inactive (latent) conformation (t(1/2) at 37 degrees C, 2.2 hours). We determined the amino acid residues responsible for the inherent instability of PAI-1, to assess whether these properties are independent and, consequently, whether the structural basis for inhibition and latency transition is different. For that purpose, a hypermutated PAI-1 library that is displayed on phage was pre-incubated for increasing periods (20 to 72 hours) at 37 degrees C, prior to a stringent selection for rapid t-PA binding. Accordingly, four rounds of phage-display selection resulted in the isolation of a stable PAI-1 variant (st-44: t(1/2) 450 hours) with 11 amino acid mutations. Backcrossing by DNA shuffling of this stable mutant with wt PAI-1 was performed to eliminate non-contributing mutations. It was shown that the combination of mutations at positions 50, 56, 61, 70, 94, 150, 222, 223, 264 and 331 increases the half-life of PAI-1 245-fold. Furthermore, within the limits of detection the stable mutants isolated are functionally indistinguishable from wild-type PAI-1 with respect to the rate of inhibition of t-PA, cleavage by t-PA, and binding to vitronectin. These stabilizing mutations constitute largely reversions to the stable "serpin consensus sequence" and are located in areas implicated in PAI-1 stability (e.g. the vitronectin-binding domain and the proximal hinge). Collectively, our data provide evidence that the structural requirements for PAI-1 loop insertion during latency transition and target proteinase inhibition can be separate