Development and Validation of a Sensitive and Specific sodB-Based Quantitative PCR Assay for Molecular Detection of Ehrlichia Species

We developed a sensitive and specific sodB-based quantitative PCR assay to detect Ehrlichia spp. The assay''s limit of detection was 5 copies/reaction, and it did not amplify nonspecific DNA. Compared with a 16S rRNA gene PCR target, the sodB target may offer an improved molecular diagnostic assay to detect Ehrlichia spp.     

Buruli ulcer. A case report]

The authors report a case of skin infection, Buruli ulcer, which is widespread in several parts of Africa: Ghana, Uganda, Ivory Coast, Senegal and most central African countries. This infection is caused by Mycobacterium ulcerans which belongs to the non-tubercular species Mycobacterium. It resembles Mycobacterium tuberculosis in colour and morphology, but differs in its speed of growth, its nutritional requirements, its capacity to produce pigments with enzymatic activities, its heat sensitivity and its resistance to anti-tubercular agents. Mycobacterium infection follows the percutaneous inoculation of the latter and appears as a painless, erythematous nodule that develops central necrosis and ulceration. Initially, the lesion appears as skin necrosis leading to the ulceration of the dermis and epidermis. The histological lesion shows a coagulative necrosis of the deep dermis and epidermis with destruction of the nerves and blood vessels; interstitial edema is also present. Healing is accompanied by a granulomatous response and the affected area is generally covered by a depressed scar. The authors initially treated the case in question using a conservative approach. A gel (Intrasite Gel) was used whose properties allowed the destruction of necrotic tissue present on the ulcer bed and the stimulation of granulation tissue formation. The layer of gel was in turn covered with a triple layer of polyurethane which enabled the humidity of the lesion to be maintained constant, thus promoting healing and acting as a barrier against external germs. This treatment enabled the skin lesion to be completely sterilised in about 30 days using new dressings every 3 days. Surgical treatment then led to complete healing after a further 20 days.     

Analytical variability in the determination of anti-double-stranded DNA antibodies: the strong need of a better definition of the old and new tests

Anti-dsDNA antibodies are a heterogeneous group of antibodies, quite specific for SLE. Their variability is related to the assay used, the immunoglobulin class secondary antibody, and the dsDNA source. The standardization of measuring anti-dsDNA antibodies is still poor and different methods yield different results. Several novel technologies were developed during the last decades that represent viable alternatives to the traditional methods such as the chemiluminescent immunoassay (CIA) and multiplex flow immunoassay (MFI). Additionally, positive results for anti-dsDNA antibodies can be detected in patients with inflammatory arthritis (IA) treated with different biologics reducing its clinical specificity for SLE. Anti-dsDNA antibody levels were evaluated in 246 patient samples: 70 SLE and 176 disease control (including 96 IA during treatment with different biologics), using three enzyme immunoassays (indirect enzyme immunoassay, Bio-Rad Laboratories; chemiluminescent immunoassay, Inova Diagnostics; multiplex flow immunoassay, Bio-Rad Laboratories) and three Crithidia luciliae immunofluorescence tests (CLIFT) (Euroimmun AG, Bio-Rad Laboratories, INOVA Diagnostics). Diagnostic performances were assessed both including and excluding the IA patients. Agreements, measured by the Cohen’s Kappa between all methods, ranged from moderate to substantial (0.47–0.68). The clinical sensitivities for the anti-dsDNA antibody tests varied from 5.7% by CLIFT A up to 33.3% provided by EIA while the clinical specificities varied from 89.8% by MFI to 98.9% provided by CLIFT B and C. Newer technologies, such as MFI and CIA, showed great potential as a diagnostic application. Significant variations among anti-dsDNA antibody assays were observed confirming the lack of standardization.     

Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis

Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1β and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-α and -β) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.     

Negative regulation of autoimmune demyelination by the inhibitory receptor CLM-1

Multiple sclerosis and its preclinical model, experimental autoimmune encephalomyelitis, are marked by perivascular inflammation and demyelination. Myeloid cells, derived from circulating progenitors, are a prominent component of the inflammatory infiltrate and are believed to directly contribute to demyelination and axonal damage. How the cytotoxic activity of these myeloid cells is regulated is poorly understood. We identify CMRF-35–like molecule-1 (CLM-1) as a negative regulator of autoimmune demyelination. CLM-1 is expressed on inflammatory myeloid cells present in demyelinating areas of the spinal cord after immunization of mice with MOG_35-55 (myelin oligodendrocyte glycoprotein) peptide. Absence of CLM-1 resulted in significantly increased nitric oxide and proinflammatory cytokine production, along with increased demyelination and worsened clinical scores, whereas T cell responses in the periphery or in the spinal cord remained unaffected. This study thus identifies CLM-1 as a negative regulator of myeloid effector cells in autoimmune demyelination.Myeloid cell–mediated destruction of myelin sheets and axons is thought to be the primary mechanism leading to loss of motor function in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE; Benveniste, 1997). The myeloid effector populations in the central nervous system (CNS) consist of resident activated microglia and blood-derived mononuclear cells including monocytes, macrophages, and DCs. A subpopulation of the infiltrating myeloid cells expresses CD11c, MHC class II, and CD86 and is often referred to as CNS DCs (Deshpande et al., 2007). These effector cells have been shown to reactivate antigen-specific T cells (Bailey et al., 2007; Deshpande et al., 2007) and are involved in epitope spreading resulting in a remitting-relapsing disease course (Miller et al., 2007). In addition to serving as antigen-presenting cells, CNS-infiltrating CD11c⁺ inflammatory myeloid cells also display T cell–independent effector functions through secretion of proinflammatory cytokines and reactive oxygen intermediates that can directly contribute to progressive demyelination and axon loss (Dogan et al., 2008; King et al., 2009).CMRF-35–like molecule-1 (CLM-1; also named MAIR-V, LMIR3, DigR2) is a member of the CD300 family of receptors, a multigene cluster of type I transmembrane glycoproteins with a single extracellular IgV domain on human chromosome 17 and mouse chromosome 11 (Márquez et al., 2007; Clark et al., 2009). Two receptors in this cluster (CLM-1 and CLM-8) contain an immunoreceptor tyrosine-based inhibition motif (ITIM) in their intracellular domains, whereas the remainder of the receptor family carries charged residues in the transmembrane region that serve to recruit signaling adapters. CLM-1, the mouse orthologue of human CD300f (Clark et al., 2009), was first described as a negative regulator of osteoclastogenesis in vitro (Chung et al., 2003). Subsequent in vitro studies have confirmed that CLM-1, or its human orthologue CD300f (also named IREM-1), serves as an inhibitory receptor in myeloid cells (Alvarez-Errico et al., 2004; Fujimoto et al., 2006; Izawa et al., 2007, 2009). One study finds that CLM-1 mediates caspase-independent cell death upon cross-linking with antibodies (Can et al., 2008). So far, a biological role in autoimmune disease has not been described. In this paper, we show that CLM-1 is expressed on iNOS- and TNF-producing inflammatory myeloid cells that invade the spinal cord after myelin oligodendrocyte glycoprotein (MOG_35-55) immunization. We further demonstrate that CLM-1 acts as a negative regulator of myeloid cell activity in vivo by suppressing the release of inflammatory cytokines and reactive oxygen species. This study thus identifies CLM-1 as a myeloid-specific negative regulator of CNS inflammation and demyelination.     

The inter-observer reading variability in anti-nuclear antibodies indirect (ANA) immunofluorescence test: A multicenter evaluation and a review of the literature

Recently there has been an increase demand for Computer-Aided Diagnosis (CAD) tools to support clinicians in the field of Indirect ImmunoFluorescence (IIF), as the novel digital imaging reading approach can help to overcome the reader subjectivity. Nevertheless, a large multicenter evaluation of the inter-observer reading variability in this field is still missing. This work fills this gap as we evaluated 556 consecutive samples, for a total of 1679 images, collected in three laboratories with IIF expertise using HEp-2 cell substrate (MBL) at 1:80 screening dilution according to conventional procedures. In each laboratory, the images were blindly classified by two experts into three intensity classes: positive, negative, and weak positive. Positive and weak positive ANA-IIF results were categorized by the predominant fluorescence pattern among six main classes. Data were pairwise analyzed and the inter-observer reading variability was measured by Cohen's kappa test, revealing a pairwise agreement little further away than substantial both for fluorescence intensity and for staining pattern recognition (k = 0.602 and k = 0.627, respectively). We also noticed that the inter-observer reading variability decreases when it is measured with respect to a gold standard classification computed on the basis of labels assigned by the three laboratories. These data show that laboratory agreement improves using digital images and comparing each single human evaluation to potential reference data, suggesting that a solid gold standard is essential to properly make use of CAD systems in routine work lab.     

Modification of actin in peritoneal macrophages after diazepam treatment

We have investigated the effect of therapeutic doses of diazepam (7 μg/mouse) on the association of actin with the macrophage cytoskeleton using cytochemical and morphological methods.

Results obtained indicated that diazepam was able to modulate the content of actin in macrophages; such an effect proved to be time-dependent. After fixation and staining for indirect immunofluorescence with actin antibody, peritoneal macrophages from mice treated for short time with diazepam, showed a fluorescent intensity increase compared to control mice. The fluorescent intensity augmented reaching peak value within 14 days of treatment. Afterwards, this value dropped below control value for mice that underwent longer treatments. In the in vitro experiments concentrations of 10^-5 M, diazepam inhibited a well cell spread and a lower amount of actin after 15 min of incubation was also revealed.

These results suggest that administration of diazepam in vivo plays a role in both the nonspecific and specific immune response, producing in the macrophages a reorganization process of microfilaments.