Follow-up of permanent hearing impairment in childhood.

Programmes for early childhood childhood hearing impairment identification allows to quickly start the appropriate hearing aid fitting and rehabilitation process; nevertheless, a large number of patients do not join the treatment program. The goal of this article is to present the results of a strategic review of the strengths, weaknesses, opportunities and threats connected with the audiologic/prosthetic/language follow-up process of children with bilateral permanent hearing impairment. Involving small children, the follow-up includes the involvement of specialised professionals of a multidisciplinary team and a complex and prolonged multi-faced management. Within the framework of the Italian Ministry of Health project CCM 2013 "Preventing Communication Disorders: a Regional Program for Early Identification, Intervention and Care of Hearing Impaired Children", the purpose of this analysis was to propose recommendations that can harmonise criteria for outcome evaluation and provide guidance on the most appropriate assessment methods to be used in the follow-up course of children with permanent hearing impairment.

Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms.

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.

Altered plasma nerve growth factor-like immunoreactivity and nerve growth factor-receptor expression in human old age.

BACKGROUND: Nerve growth factor (NGF), discovered because of its action on cells in the peripheral and the central nervous system, is now known to act also on immune cells in developing and adult subjects. Whether peripheral lymphocytes of aged subjects are responsive to this molecule is less clear. OBJECTIVE: Aim of our study was to investigate whether or not plasma NGF undergoes any significant changes during aging and whether or not NGF receptors on peripheral lymphocytes are affected in old subjects. METHODS: To address these questions, we used RT-PCR, immunohistochemistry, and immunoenzymatic analysis to evaluate the expression of NGF receptors in human peripheral lymphocytes and NGF-like immunoreactivity. RESULTS: These results revealed a low amount of NGF in the plasma of old subjects associated with a reduced expression of mRNA for NGF receptor trkA in peripheral lymphocytes. They also showed elevated levels of circulating interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) in the bloodstream of adult and aged subjects. CONCLUSION: Our data indicate that peripheral lymphocytes are targets for circulating NGF and suggest that changes in NGF-receptor expression on lymphocytes might be potential marker for aging lymphocytes.

Nerve growth factor in neurological and non-neurological diseases: basic findings and emerging pharmacological prospectives.

Nerve growth factor (NGF) is known to be essential for the survival of peripheral and brain neurons, and according to more recent studies also for a variety of cells localized in the immune system. Basic and preclinical findings published in the last 15-20 years have prospected the hypothesis that NGF can be pharmaceutically useful for promoting healing in certain peripheral and central neurological insults. We have recently provided evidence that NGF applied topically, has a therapeutic potentiality for human corneal and pressure ulcers, and more recently in vasculitis induced by rheumatoid arthritis. This review will summarize previous and ongoing evidence supporting the role of NGF in the nervous and immune system and discuss NGF potentiality as a pharmacological tool for basic and clinical studies.

Human monocyte/macrophages activate by exposure to LPS overexpress NGF and NGF receptors.

Monocyte/macrophages (M/M) represent the main cellular component of the immune system involved in the inflammatory response. In the present study we investigate whether NGF is produced by M/M and is involved in this event. The results show that unstimulated human M/M produce NGF and its synthesis is stimulated by LPS. The increase of NGF is associated with enhanced expression of high affinity NGF receptor on M/M and with no changes of low affinity NGF receptors (p75). The neutralization of endogenous NGF by NGF antibody in LPS-activated M/M, leads to overexpression of p75 receptor causing apoptosis. These findings provide new insight in the mechanisms governing monocyte survival in the inflamed tissue, representing a crucial aspect of host defence and maintenance of homeostasis.

Altered levels of neuropeptides characterize the brain of lupus prone mice.

It has been reported that more than 50% of lupus patients show various forms of neurological deficits including impaired cognitive functions and psychiatric disorders. Using an animal model of lupus we investigated the production of neuropeptides in the brain of NZB/W F1 female hybrid mice and its parental strain NZB and NZW. Our results indicate that the alteration in learning and memory described in lupus mice are paralleled by a decrease in calcitonin gene-related peptide, substance P and neuropeptide Y (NPY) levels in the hippocampus and a significant decrease of NPY in the cortex. These findings are interesting in the light of previously reported results suggesting that these neuropeptides can play an important role in cognitive functions. We also observed a decrease of NPY and vasoactive intestinal polypeptide levels in the hypothalamus of lupus prone mice and these changes may be related to the disregulation of the hypothalamus-pituitary-adrenal axis observed in lupus prone mice.

Development of systemic lupus erythematosus in mice is associated with alteration of neuropeptide concentrations in inflamed kidneys and immunoregulatory organs.

In the present study we used a well-characterised model of murine lupus, the female NZB/W hybrid, to study the possible involvement of neuropeptides in the pathogenesis of systemic lupus erythematosus (SLE). Analysis of neuropeptides with a possible role in inflammation showed that substance P (SP) calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) are present in increased quantities in the inflamed kidneys of SLE mice, confirming their involvement in local inflammation, while there is a general reduction in the peptide concentrations in the lymphoid organs of lupus mice, except for NPY. Our results suggest that the altered neuropeptide concentrations observed in the SLE lymphoid organs may be partly responsible for the altered immune response and contribute to the development of autoimmune diseases.

Human CD4+ T cell clones produce and release nerve growth factor and express high-affinity nerve growth factor receptors.

BACKGROUND: Increasing evidence shows that nerve growth factor (NGF) plays a role in the complex and fascinating linkage between the nervous and the immune systems due to its ability to modulate functions of several inflammatory cells. OBJECTIVE: To investigate NGF receptor expression and NGF production and release by human CD4+ cells clones, which have primary relevance in modulating inflammatory events through their different subsets of functional phenotypes. METHODS: The expression of NGF and a transmembrane tyrosine kinase (TrkA) was evaluated by immunohistochemistry and flow cytometry analysis in five T(H0), six T(H1), and five T(H2) cell clones derived from human circulating mononuclear blood cells. Moreover, the amount of NGF protein was assessed by measuring the NGF levels in culture supernatants of the T cell clones before stimulation and 48 hours after phytohemagglutinin (PHA) activation by use of an immunoenzymatic assay. RESULTS: Our data have shown that in unstimulated conditions, human CD4+ T cell clones express both immunoreactivity for NGF and the TrkA NGF receptor irrespective of their cytokine profile. Moreover, T(H1) and T(H2) clones, but not T(H0) clones, secrete NGF in basal conditions. PHA activation induces NGF secretion in T(H0) clones and a significant increase of NGF levels in T(H2) (p < 0.05), but not in T(H1) culture supernatants. CONCLUSIONS: Results obtained represent the first evidence of TrkA expression and NGF production and release in human CD4+ cell clones and suggest a possible functional role of NGF in modulating the immune and inflammatory network.

The expanding role of nerve growth factor: from neurotrophic activity to immunologic diseases.

Numerous studies published in the last 10-15 years have shown that nerve growth factor (NGF), a polypeptide originally discovered in connection with its neurotrophic activity, also acts on cells of the immune system. NGF has been found in various immune organs including the spleen, lymph nodes, and thymus, and cells such as mast cells, eosinophils, and B and T cells. The circulating levels of NGF increase in inflammatory responses, in various autoimmune diseases, in parasitic infections, and in allergic diseases. Stress-related events both in animal models and in man also result in an increase of NGF, suggesting that this molecule is involved in neuroendocrine functions. The rapid release of NGF is part of an alerting signal in response to either psychologically stressful or anxiogenic conditions in response to homeostatic alteration. Thus, the inflammation and stress-induced increase in NGF might alone or in association with other biologic mediators induce the activation of immune cells during immunologic insults. A clearer understanding of the role of NGF in these events may be useful to identify the mechanisms implicated in certain neuroimmune and immune dysfunctions.

Presence of nerve growth factor in the thymus of prenatal, postnatal and pregnant rats.

The presence of Nerve Growth Factor (NGF) and the expression of low- and high-affinity NGF receptor were investigated in prenatal, postnatal and pregnant rats. Using ELISA and immunohistochemistry it was found that both NGF and its receptors are present in the medulla of the thymus and are more strongly expressed in pre- and early postnatal life and nearly absent in adult and ageing rats. It was also found that during pregnancy, which is characterised by an involution of the cortex and hypertrophy of the medulla, the level of NGF in the thymus increases. The present study showed not only that NGF is produced in a specific compartment of the thymus, the medulla, but that its synthesis declines with age, following thymus involution. These results suggest that NGF may be critically involved in the proliferation and differentiation of thymic cells.

Nerve growth factor is an autocrine survival factor for memory B lymphocytes.

Production of nerve growth factor (NGF) was assessed in cultures of human T and B lymphocytes and macrophages. NGF was constitutively produced by B cells only, which also expressed surface p140trk-A and p75NGFR molecules and hence efficiently bound and internalized the cytokine. Neutralization of endogenous NGF caused disappearance of Bcl-2 protein and apoptotic death of resting lymphocytes bearing surface IgG or IgA, a population comprising memory cells, while surface IgM/IgD "virgin" B lymphocytes were not affected. In vivo administration of neutralizing anti-NGF antibodies caused strong reduction in the titer of specific IgG in mice immunized with tetanus toxoid, nitrophenol, or arsonate and reduced numbers of surface IgG or IgA B lymphocytes. Thus, NGF is an autocrine survival factor for memory B lymphocytes.

Modification of lymphoid and brain nerve growth factor levels in systemic lupus erythematosus mice.

In the present work we investigated the production of nerve growth factor (NGF) in the brain and peripheral tissues of female NZB/W F1 mice, a well characterized model of murine lupus. Our results indicate that while no significant difference in the NGF content was observed in the sera and tissues of NZB/W mice and its parental strains during the first months of life, the levels of circulating NGF and the NGF content in the kidneys significantly increase in the autoimmune mice during the development of the disease. The NGF-producing brain regions showed a decrease in NGF concentration in 8 month-old NZB/W mice. Moreover, we found a modification of the NGF concentration in the spleens of autoimmune mice at 5 and 8 months. Our data support the hypothesis of a correlation between NGF and the inflammatory state of systemic lupus erythematosus (SLE) and indicate that NGF could have a role in the pathogenesis of this disease.

Nerve growth factor stimulates production of neuropeptide Y in human lymphocytes.

The production of neuropeptide Y (NPY) in lymphocytes obtained from human tonsils was investigated using radioimmunoassay. While unstimulated lymphocytes did not produce detectable amounts of NPY, NPY synthesis was induced after cell activation. Our results show that the addition of nerve growth factor (NGF) to unstimulated lymphocytes has an effect similar to that of mitogens, both leading to production of NPY. The study of purified B and T cells confirmed that only activated cells are able to synthesize NPY. The stimulatory effect of NGF on NPY production is not a common characteristic of all lymphocytes: only unstimulated T cells respond to NGF by synthesizing NPY. No such effects is seen in purified B cells.

Monosodium glutamate increases NGF and NPY concentrations in rat hypothalamus and pituitary.

The effects of MSG treatment on NGF and NPY levels were analysed in the hypothalamus, pituitary, adrenal, thyroid and testis of adult rats. Daily i.v. injections of MSG (1 g kg-1 for 1 week) induced an increase of NGF in the hypothalamus (control (C) = 378 +/- 54; saline (S) = 369 +/- 36; MSG = 479 +/- 35 pg g-1 tissue; p < 0.001) and pituitary (C = 310 +/- 34; S = 376 +/- 114; MSG = 576 +/- 98 pg g-1 tissue; p < 0.01). Hypothalamic and pituitary NPY concentrations were also altered in the MSG-treated rats. Compared with saline-treated rats, the NPY concentration increased by 43% in the hypothalamus and 37.5% in the pituitary of MSG-treated rats. No significant changes in NGF and NPY content were found in the adrenal or thyroid of treated animals. These results suggest that hypothalamic and pituitary NGF and NPY levels may be involved in the control of neuroendocrine functions that are affected by MSG treatment.

Increased plasma levels of nerve growth factor in vernal keratoconjunctivitis and relationship to conjunctival mast cells.

PURPOSE: To evaluate the nerve growth factor (NGF) plasma concentration in patients with vernal keratoconjunctivitis and to correlate it with the histopathology and immunopathology of the disease. METHODS: An immunoenzymatic assay was performed to measure NGF plasma levels in patients with vernal keratoconjunctivitis and in healthy matched controls. A competitive radioimmunoassay was used to detect eosinophil cationic protein (ECP) and total specific immunoglobulin E (IgE) serum levels. Histologic evaluation was performed in tarsal and bulbar conjunctival biopsies. RESULTS: Plasma levels of NGF were significantly higher (P < 0.001) in patients with vernal keratoconjunctivitis (mean = 8224.47 +/- 7802.53 pg/ml; median = 121 pg/ml) than in controls (mean = 51.68 +/- 5.94 pg/ml; median 42.5 pg/ml). Conjunctival tissue showed a significant increase of mast cells, eosinophils, and lymphocytes in vernal keratoconjunctivitis. A significant correlation was observed between plasma levels of NGF and the number of mast cells in the tarsal conjunctiva (Cc = 0.81; P < 0.005) and bulbar conjunctiva (Cc = 0.77; P < 0.01) of patients with vernal keratoconjunctivitis. No correlation was found between NGF plasma levels and total IgE serum levels in patients with vernal keratoconjunctivitis; NGF plasma levels were inversely related to the number of circulating eosinophils (Cc = -0.61; P < 0.05) and to the increased serum levels of ECP (Cc = -0.71; P < 0.02). CONCLUSIONS: These data represent the first reported evidence of increased NGF plasma levels in an allergic human disease and suggest a possible relationship between this neurotrophic polypeptide and inflammatory cells in vernal keratoconjunctivitis.

Emotional stress induced by parachute jumping enhances blood nerve growth factor levels and the distribution of nerve growth factor receptors in lymphocytes.

We examined the plasma nerve growth factor (NGF) level and the distribution of NGF receptors in peripheral lymphocytes of young soldiers (mean age, 20-24 yr) experiencing the thrill of a novice about to make their first parachute jumps. Blood was collected from soldiers who knew they were selected to jump (n = 26), as well as from soldiers who knew they were not selected (n = 17, controls). The former group was sampled the evening before the jump and 20 min after landing. Compared with controls, NGF levels increased 84% in prejump and 107% in postjump sampling. Our studies also showed that the increase of NGF levels preceded the increase of plasma cortisol and adrenocorticotropic hormone. No changes in the baseline levels of circulating interleukin 1 beta or tumor necrosis factor were found, suggesting that the increased levels of NGF were not correlated with change in these cytokines. Moreover, immunofluorescence analysis demonstrated that parachuting stress enhances the distribution of low-affinity p75LNGFR and high-affinity p140trkA NGF receptors in circulating peripheral blood mononuclear cells. These observations suggest that the release of NGF might be involved in the activation of cells of the immune system and is most probably associated with homeostatic adaptive mechanisms, as previously shown for stressed rodents.

Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes.

Keratinocytes, a key cellular component both for homeostasis and pathophysiologic processes of the skin, secrete a number of cytokines and are stimulated by several growth factors. Nerve growth factor (NGF) is synthesized in the skin and basal keratinocytes express the low-affinity nerve growth factor receptor (NGF-R). We present evidence that normal human keratinocytes in culture express the low- and the high-affinity NGF-R both at the mRNA level, as determined by reverse-transcription polymerase chain reaction and at the protein level, as shown by cytofluorimetric analysis. NGF significantly stimulates the proliferation of normal human keratinocytes in culture in a dose-dependent manner. This effect can be prevented by the addition of both an anti-NGF neutralizing antibody and a high-affinity NGF-R (trk) specific inhibitor, the natural alkaloid K252a. By contrast, keratinocyte proliferation is not inhibited by an anti-low-affinity NGF-R monoclonal antibody, thus suggesting that NGF effect on human keratinocytes is mediated by the high-affinity NGF-R. Moreover, NGF mRNA is expressed in normal human keratinocytes and NGF is secreted by keratinocytes in increasing amounts during growth, as detected by enzyme-linked immunosorbent assay. These results suggest that NGF could act as a cytokine in human skin and take part in disorders of keratinocyte proliferation.

NGF is released into plasma during human pregnancy: an oxytocin-mediated response?

The presence of biologically active nerve growth factor (NGF) in the peripheral circulation of women during pregnancy, labour and lactation was investigated. Using a sensitive immunoenzymatic assay (ELISA), we found an approximately five-fold increase in plasma NGF levels during labour and lactation compared with the concentrations found at the term of gestation or in control healthy women. Since labour and lactation are characterized by activation of the hypothalamo-pituitary-adrenal axis and by high plasma levels of the neurohypophyseal hormone oxytocin, and since the intravenous injection of oxytocin in female rats causes a 176% increase in the hypothalamic levels of NGF, it is possible that the increased amount of circulating NGF is correlated with one or both of these events.

NGF retards apoptosis in chick embryo bursal cell in vitro.

Recent studies have demonstrated that the action of nerve growth factor (NGF) is not restricted to neuronal cells but also affects cells of the immune system. In a previous work on the effect of NGF on the chick embryo bursa of Fabricius both in vivo and in vitro, we observed that NGF prolongs bursal cell survival in vitro. In the present study we report that the increase of viable cells in NGF-treated cultures is not due to a proliferative effect of NGF on bursal cells but to a reduction of cell mortality. The morphological analysis revealed that bursal cells in cultures die by apoptosis, which was also shown by the typical pattern of DNA fragmentation, a hallmark of this cell death process. It is concluded that NGF, with an action similar to that described in sympathetic neurons and PC12, could retard bursal cell death by influencing apoptosis.