Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA.

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.     

Toll-like receptors 3 and 4 are expressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) are multipotent, nonhemopoietic progenitors that also possess regulatory activity on immune effector cells through different mechanisms. We demonstrate that human BM-derived MSCs expressed high levels of Toll-like receptors (TLRs) 3 and 4, which are both functional, as shown by the ability of their ligands to induce nuclear factor kappaB (NF-kappaB) activity, as well as the production of interleukin (IL)-6, IL-8, and CXCL10. Of note, ligation of TLR3 and TLR4 on MSCs also inhibited the ability of these cells to suppress the proliferation of T cells, without influencing their immunophenotype or differentiation potential. The TLR triggering effects appeared to be related to the impairment of MSC signaling to Notch receptors in T cells. Indeed, MSCs expressed the Notch ligand Jagged-1, and TLR3 or TLR4 ligation resulted in its strong downregulation. Moreover, anti-Jagged-1 neutralizing antibody and N[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of Notch signaling, hampered the suppressive activity of MSCs on T-cell proliferation. These data suggest that TLR3 and TLR4 expression on MSCs may provide an effective mechanism to block the immunosuppressive activity of MSCs and therefore to restore an efficient T-cell response in the course of dangerous infections, such as those sustained by double-stranded RNA viruses or Gram-negative bacteria, respectively.     

Alport syndrome: impact of digenic inheritance in patients management

Alport syndrome (ATS) is a genetically heterogeneous nephropathy with considerable phenotypic variability and different transmission patterns, including monogenic (X‐linked/autosomal) and digenic inheritance (DI). Here we present a new series of families with DI and we discuss the consequences for genetic counseling and risk assessment. Out of five families harboring variants in more than one COL4 gene detected by next generation sequencing (NGS), minigene‐splicing assay allowed us to identify four as true digenic. Two families showed COL4A3/A4 mutations in cis, mimicking an autosomal dominant inheritance with a more severe phenotype and one showed COL4A3/A4 mutations in trans, mimicking an autosomal recessive inheritance with a less severe phenotype. In a fourth family, a de novo mutation (COL4A5) combined with an inherited mutation (COL4A3) triggered a more severe phenotype. A fifth family, predicted digenic on the basis of silico tools, rather showed monogenic X‐linked inheritance due to a hypomorphic mutation, in accordance with a milder phenotype. In conclusion, this study highlights the impact of DI in ATS and explains the associated atypical presentations. More complex inheritance should be therefore considered when reviewing prognosis and recurrence risks. On the other side, these findings emphasize the importance to accompany NGS with splicing assays in order to avoid erroneous identification of at risk members.     

The purinergic P2×7 receptor is expressed on monocytes in Behçet's disease and is modulated by TNF‐α

The P2×7 receptor (P2×7r) is expressed in innate immune cells (e.g. monocyte/macrophages), playing a key role in IL‐1β release. Since innate immune activation and IL‐1β release seem to be implicated in Behçet's disease (BD), a systemic immune‐inflammatory disorder of unknown origin, we hypothesized that P2×7r is involved in the pathogenesis of the disease. Monocytes were isolated from 18 BD patients and 17 healthy matched controls. In BD monocytes, an increased P2×7r expression and Ca²⁺ permeability induced by the selective P2×7r agonist 2′‐3′‐O‐(4‐benzoylbenzoyl)ATP (BzATP) was observed. Moreover, IL‐1β release from LPS‐primed monocytes stimulated with BzATP was markedly higher in BD patients than in controls. TNF‐α‐incubated monocytes from healthy subjects almost reproduced the findings observed in BD patients, as demonstrated by the increase in P2×7r expression and BzATP‐induced Ca²⁺ intake. Our results provide evidence that in BD monocytes both the expression and function of the P2×7r are increased compared with healthy controls, as the possible result, at least in part, of a positive modulating effect of TNF‐α on the receptor. These data indicate P2×7r as a new potential therapeutic target for the control of BD, further supporting the rationale for the use of anti‐TNF‐α drugs in the treatment of the disease.     

High-affinity monomeric 67-kd laminin receptors and prognosis in pancreatic endocrine tumours

Cell-surface high-affinity monomeric 67-kD laminin receptors have been proposed to promote the invasion and metastasis of a variety of tumours, but there are, as yet, no data regarding the expression of these molecules in pancreatic endocrine tumours (PETs). The prognosis of these very rare tumours is problematic and the only irrefutable evidence of their malignancy still continues to be the occurrence of local invasion and metastases. In this retrospective investigation, 34 functioning and 48 non-functioning sporadic PETs were evaluated for the expression of the MLuC5 monoclonal antibody, which specifically recognizes the 67-kD laminin receptors. Laminin receptors were found in 42/82 cases (51 per cent) and their expression was associated with metastatic disease (P<0·001), high proliferative activity expressed by a Ki-67 index above 5·0 per cent (P<0·001), absence of progesterone receptors (P=0·013), immunoreactivity for hormones other than insulin (P<0·001), a tumour diameter more than 3·0 cm (P=0·001), and a fatal clinical outcome (P<0·001). Laminin receptors were also expressed by most metastatic foci and all intravascular emboli of tumour cells. Positivity for laminin receptors was associated with shorter survival in functioning (P=0·026) and non-functioning (P=0·042) tumours, as well as in the whole series of pancreatic endocrine tumours (P<0·001). On multivariate analysis, laminin receptor expression was not an independent prognostic factor, while a Ki-67 index above 5·0 per cent was the most powerful predictor of survival. However, the association of laminin receptor expression and Ki-67 index could identify a group of malignant PETs with low proliferative activity characterized by an intermediate prognosis. In conclusion, these data suggest that monomeric laminin receptors may play a role in the invasion and metastasis of PETs and that their expression may be an additional prognostic factor, along with proliferative activity. © 1997 by John Wiley & Sons, Ltd.     

Reduced progression of atherosclerosis in apolipoprotein E-deficient mice treated with lacidipine is associated with a decreased susceptibility of low-density lipoprotein to oxidation

A study has been carried out in the apolipoprotein (apo) E-deficient mouse to investigate the activity of lacidipine (a calcium antagonist with antioxidant properties) in inhibiting the development of atherosclerotic lesions; of particular interest were changes in the susceptibility of low-density lipoproteins (LDL) to oxidation. Mice receiving a Western-type diet to accelerate the development of atherosclerosis were treated orally with vehicle or lacidipine at 3 or 10 mg/kg/day for 8 weeks. Lacidipine treatment (at 3 or 10 mg/kg) had no effect on the plasma lipid profile. However, a significant (P < 0.01) dose-related reduction of 43 and 50% of the aortic lesion area in respect to vehicle-treated mice was observed. Moreover, the resistance of mouse plasma LDL to undergo lipid peroxidation was significantly (P < 0.01) increased in apo E-deficient mice treated with lacidipine. The native LDL-like particle, derived from apo E-deficient mice treated with lacidipine, contained significantly lower concentrations of malonyldialdehyde than the vehicle-treated control group (P < 0.01). After exposure to human umbilical vein endothelial cells, LDL-like particle vitamin E levels (expressed as area under the curve; AUC), were significantly higher (P < 0.01) in both the 3 and 10 mg/kg lacidipine-treated groups, in comparison with the vehicle-treated control animals. We conclude that lacidipine reduced the extent of the atherosclerotic area in hypercholesterolaemic apo E-deficient mice, and that this reduction may be associated with the capacity of the drug to decrease the susceptibility of LDL to oxidation.     

Clinical and molecular genetics of patients with the Carney-Stratakis syndrome and germline mutations of the genes coding for the succinate dehydrogenase subunits SDHB, SDHC, and SDHD

Gastrointestinal stromal tumors (GISTs) may be caused by germline mutations of the KIT and platelet-derived growth factor receptor-alpha (PDGFRA) genes and treated by Imatinib mesylate (STI571) or other protein tyrosine kinase inhibitors. However, not all GISTs harbor these genetic defects and several do not respond to STI571 suggesting that other molecular mechanisms may be implicated in GIST pathogenesis. In a subset of patients with GISTs, the lesions are associated with paragangliomas; the condition is familial and transmitted as an autosomal-dominant trait. We investigated 11 patients with the dyad of 'paraganglioma and gastric stromal sarcoma'; in eight (from seven unrelated families), the GISTs were caused by germline mutations of the genes encoding subunits B, C, or D (the SDHB, SDHC and SDHD genes, respectively). In this report, we present the molecular effects of these mutations on these genes and the clinical information on the patients. We conclude that succinate dehydrogenase deficiency may be the cause of a subgroup of GISTs and this offers a therapeutic target for GISTs that may not respond to STI571 and its analogs.     

Overflow movements and white matter abnormalities in ADHD

Multiple motor abnormalities have been identified in some children with Attention Deficit/Hyperactivity Disorder (ADHD). These include persistence of overflow movements, impaired timing of motor responses and deficits in fine motor abilities. Motor overflow is defined as co-movement of body parts not specifically needed to efficiently complete a task. The presence of age-inappropriate overflow may reflect immaturity of the cortical systems involved in automatic motor inhibition. Theories on overflow movements consistently implicate impairments in white matter (WM) tracts, including the corpus callosum. WM connections might be altered selectively in brain networks and thus influence motor behaviours. We reviewed the scientific contributions on overflow movements and WM abnormalities in ADHD. They suggest that WM abnormalities in motor/premotor circuits, which are important for motor response inhibition, might be responsible for overflow movements in patients with ADHD.