Pain in individuals with RASopathies: Prevalence and clinical characterization in a sample of 80 affected patients

Pain in individuals with RASopathies is a neglected topic in literature. In this article, we assessed prevalence and profile of pain in a sample of 80 individuals affected by RASopathies. The study sample included individuals with Noonan syndrome (N = 42), Costello syndrome (N = 17), and cardio‐facio‐cutaneous syndrome (N = 21). A set of standardized questionnaires and scales were administered (VAS/numeric scale, r‐FLACC, Wang‐Baker scale, NPSI, BPI, NCCPC‐R) to detect and characterize acute and chronic pain and to study the influence of pain on quality of life (PEDs‐QL, SF‐36) and sleeping patterns (SDSC); revision of past medical history and multisystemic evaluation was provided. Available clinical data were correlated to the presence of pain. High prevalence of acute (44%) and chronic (61%) pain was documented in the examined sample. Due to age and intellectual disability, acute pain was localized in 18/35 individuals and chronic pain in 33/49. Muscle‐skeletal and abdominal pain was more frequently reported. The intensity of acute and chronic pain interfered with daily activities in 1/3 of the sample. Pain negatively impacted on QoL and sleeping patterns. This work documents that pain is highly prevalent in RASopathies. Future studies including subjective and objective measures of pain are required to discriminate a somatosensory abnormality from an abnormal elaboration of painful stimuli at a central level.     

Effect of sulforaphane on micronucleus induction in cultured human lymphocytes by four different mutagens

Isothiocyanates (ITCs) are commonly found in cruciferous vegetables. A variety of biological activities have been ascribed to ITCs, such as inhibition of cytochrome P450 enzymes and induction of phase II enzymes in animal models. ITCs are also able to block cell-cycle progression and induce apoptosis in human cancer cells in vitro. In this study, we evaluated the ability of the ITC sulforaphane to protect cultured human lymphocytes from micronucleus (MN) induction by four different mutagens: ethyl methanesulfonate (EMS), vincristrine (VIN), H(2)O(2) and mitomycin C (MMC). To understand the mechanisms of action of sulforaphane, the cultures were treated with the compound before, during and after treatment with the mutagens; in addition, the cultures were evaluated for the induction of apoptosis. Up to 10 microM, sulforaphane was non-genotoxic by itself, while 30 microM sulforaphane reduced the replicative index of the cells by more than 60%. Moreover, 1-10 microM sulforaphane reduced the MN frequency induced by EMS, VIN, H(2)O(2) and MMC in at least one of the treatment protocols; it had no effect on H(2)O(2)-MN induction in the post-treatment protocol, and it increased MN induction by MMC in the pre-treatment protocol. Apoptosis was produced in the cultures treated with sulforaphane alone. The fraction of apoptotic cells was increased after co- or post-treatment with sulforaphane and EMS and MMC, suggesting that sulforaphane-mediated apoptosis may remove highly damaged cells induced by these agents. Other mechanisms are involved in the anti-genotoxic activity of sulforaphane against VIN and H(2)O(2). Taken together, our findings indicate that under certain conditions sulforaphane possesses anti-genotoxic activity in vitro and that further studies are warranted to characterize this property in vivo.     

Sequence distribution of poly(ether-sulfone)/poly(ether-ketone) copolymers by mass spectrometry and 13C NMR

The sequence distributions of four poly(ether-sulfone)/poly(ether-ketone) (PES/PEK) copolymer samples were determined by means of fast atom bombardment mass spectrometry (FAB-MS) and by ¹³C NMR spectroscopy. The controlled partial degradation of the copolymer chains to produce oligomers suitable of FAB-MS analysis was performed with sodium methoxide in dimethyl sulfoxide solution at 130°C, and the experimental conditions were optimized. The sequential arrangements of ether-sulfone/ether-ketone units present in these materials were estimated by a best-fit minimization method using the MACO4 computer program which compares the experimental FAB-MS spectral intensities with theoretical intensities. Random PES/PEK copolymer samples showed quite the same sequence arrangements as expected from monomer feed-ratios used in the syntheses. Instead, a PES/PEK copolymer sample expected to be exactly alternating (from the synthesis procedure) showed 44% of random sequences owing to the transetherification reaction which occurred in the synthesis. The results of sequential analysis obtained from ¹³C NMR data compare well with the data obtained by the FAB-MS analysis.     

A B7-1-transfected human melanoma line stimulates proliferation and cytotoxicity of autologous and allogeneic lymphocytes

B7 co-stimulation is necessary to activate resting T cells upon antigen recognition by the T cell receptor. To see whether expression of B7 may render human melanoma cells able to stimulate T cells, a cloned melanoma line (Me1B6), which did not express B7-1, was transfected with the human B7-1 gene. In proliferation assays, B7-1 transfected cells (Me1B6/B7) showed greater stimulatory activity of allogeneic and autologous peripheral blood lymphocytes (PBL) compared to parental, non-transfected tumor cells. This effect was also seen when allogeneic CD8⁺ and CD4⁺ subpopulations were used as effectors. In these studies, activation of lymphocytes was B7-1-dependent and HLA classes I and II mediated. The higher proliferation correlated with an increased lytic activity by PBL stimulated with B7-1⁺ tumor cells against the untransfected Me1B6. Furthermore, PBL from a metastatic melanoma patient stimulated by Me1B6/B7 developed an higher lytic activity not only against Me1B6 but also against their autologous, B7-1^? tumor. Finally, after Me1B6/B7 stimulation, PBL released interleukin (IL)-2 and interferon-γ, but not IL-4, suggesting a Th1-mediated response. These data support the use of B7-1 transfected melanoma cells in the therapeutic vaccination of melanoma patients.     

Early increase precedes a depletion of VIP and PGP-9.5 in the skin of insulin-dependent diabetics—correlation between quantitative immunohistochemistry and clinical assessment of peripheral neuropathy

Diabetic neuropathy affects both sensory and autonomic peripheral nerve fibres. Vasoactive intestinal polypeptide (VIP) is present in autonomic fibres which modulate sweat secretion, while calcitonin gene-related peptide (CGRP) is localized to cutaneous sensory fibres. In this study, immunohistochemistry and image analysis were used to assess changes of VIP and CGRP, and of the pan-neuronal marker protein gene-product (PGP)-9.5, in skin biopsies of 18 patients affected by type 1 diabetes (age range 18–46 years) and from seven aged-matched controls. Patients were divided into three groups: group 1 (n=6), with diabetes for 6 months to 3 years; group 2 (n=5), with the disease for 5–10 years; and group 3 (n=7), with diabetes for more than 10 years. VIP immunoreactivity (IR) and PGP-9.5-IR were significantly reduced around sweat glands (P <0.005) in groups 2 and 3. Epidermal CGRP-IR and PGP-9.5-IR were significantly reduced in group 3 (P <0.05). Twenty-eight per cent (5/18) of all patients showed high VIP-IR around sweat glands (>95 per cent confidence limits of controls) and all of these patients had diabetes for less than 3 years. Conversely, 55 per cent (10/18) of patients had low VIP-IR (<5 per cent confidence limit of controls). The latter, compared with the former, showed a significantly longer duration of diabetes (Fisher exact test P=0·002), presence of clinical autonomic neuropathy (Fisher exact test P=0.04), and a reduced sural nerve conduction velocity (Fisher exact test P=0.04). These results suggest that quantitative immunohistochemical analysis of peptide-containing cutaneous nerves allows an objective evaluation of nerve fibre alterations at early stages of diabetes than is currently possible with neurophysiological functional tests.     

Effect of hypothermia on renal sodium reabsorption

Summary The relationship between sodium reabsorption and oxygen consumption was studied in an isolated rabbit kidney preparation perfused with blood at 37, 28 and 19° C. When the temperature was lowered from 37° C to 28° C and to 19°C the rate of oxygen consumption and of the maximal P.A.H. excretion (Tm P.A.H.) decreased more than that of sodium reabsorption.TheQ ₁₀ for sodium reabsorption is about 1.8, while that for maximal P.A.H. excretion is 2.5. Some hypothesis on the possible mechanisms of the lowQ ₁₀ of the Na⁺ reabsorption are forwarded.Preliminary reports have been published [Boll. Soc. Ital. Biol. Sper.43, 1019–1023 (1966) and44, 1784–1787 (1967);45, 860–862 (1969) and45, 863–865 (1969)].     

Extracellular HIV-1 Tat protein activates phosphatidylinositol 3- and Akt/PKB kinases in CD4+ T lymphoblastoid Jurkat cells

The biological basis for the pleiotropic activity of extracellular human immunodeficiency virus (HIV)-1 Tat protein on lymphoid T cell survival is not well understood. We have here demonstrated that the addition in culture of 0.1–10 nM Tat protein to 36-h serum-starved lymphoblastoid Jurkat T cells rapidly stimulates the catalytic activity of phosphatidylinositol 3-kinase (PI 3-K). The peak of activation was observed 30 min after Tat addition. Extracellular Tat also stimulated the catalytic activity of the Akt/PKB kinase, a major target of PI 3-K lipid products. Pretreatment of serum-starved Jurkat cells with 100 nM wortmannin (WT) or 10 μM LY294002, two unrelated pharmacological inhibitors of PI 3-K, markedly suppressed the catalytic activity of both PI 3-K and Akt/PKB in Jurkat cells. Moreover, at low concentrations (0.1–1 nM), extracellular Tat showed a small but reproducible protection of Jurkat cells from apoptosis induced by serum deprivation (p < 0.05), while the combination of Tat plus 100 nM WT significantly (p < 0.05) increased the percentage of apoptosis with respect to cells left untreated or treated with Tat alone. Taken together, these data suggest that the anti-apoptotic activity of low concentrations of Tat protein on Jurkat cells is mediated by a PI 3-kinase/Akt pathway.     

TS/A: a new metastasizing cell line from a BALB/c spontaneous mammary adenocarcinoma

A metastasizing mouse cell line (TS/A), originated from a mammary adenocarcinoma which arose spontaneously in a BALB/c female retired breeder, has been established in vitro. It displayed a remarkable morphologic heterogeneity, which is evident in plastic adherent cultures (cell types ranging from epithelial-like to fibroblast-like) as well as in semi-solid agar cultures. The TS/A line exhibited the presence of specific cytoplasmic estradiol receptor, with a binding activity of 16 fmoles/mg cytosol protein. The in vivo growth pattern was as follows: (1) a s.c. inoculum of 105 cells caused a 100 per cent tumor take and kill in syngeneic animals; mean survival time was 54 + 1 days; (2) it did not show significant transplant immunogenicity in syngeneic animals; (3) it was able to give rise to both spontaneous lung metastases and artificial lung colonies; (4) it had a high capacity to grow in H-2 matched, minor histocompatibility antigen incompatible hosts (106 cells killed 100 per cent DBA/2 mice in 58 + 2 days). This line of spontaneous mammary tumor cells is proposed as a useful model for studies on the heterogeneity of the neoplastic population in relation to metastatic spread, on tumor immunogenicity, and on therapy of mammary neoplasia.     

Osteocyte ultrastructure in renal osteodystrophy

Summary The ultrastructure of the osteocyte has been studied in 80 needle biopsies from the iliac crest of uremic subjects with renal osteodystrophy.Different types of osteocytes were present in the osseous trabeculae. Those recognizable in completely uncalcified osteoid tissue looked like normal osteocytes, even though the matrix was not mineralized. Those present in hypomineralized areas showed enlarged and irregular lacunae when examined under the light microscope; under the electron microscope these osteolytic-like changes were not evident and were found to have been produced by defective calcification of the perilacunar matrix. Osteocytes placed in matrix whose mineralization was normal were often surrounded by a border of crystals protruding side-to-side from the bone matrix into the lacunar space. Other osteocytes were placed in unusually wide lacunae. They showed evidence of osteolytic activity, chiefly consisting of irregularity of the lacunar wall, presence of flocculent, granular and filamentous material in the pericellular space, and calcification of mitochondria. Degenerating and degenerate osteocytes were also recognizable.