Increasing pathomorphism of pulmonary tuberculosis: an observational study of slow clinical, microbiological and imaging response of lung tuberculosis to specific treatment. Which role for linezolid?

During recent years, a progressive emerging of tuberculosis occurred, related to the overall increased age of general population, primary and secondary (iatrogenic) immunodeficiencies, the availability of invasive procedures, surgical interventions and intensive care supports, bone marrow and solid organ transplantation, and especially the recent immigration flows of people often coming from areas endemic for tuberculosis, and living with evident social-economical disadvantages, and with a reduced access to health care facilities. Since January 2006, at our reference centre we followed 81 consecutive cases of pulmonary tuberculosis, with 65 of them which remained evaluable for the absence of extrapulmonary complications, and a continuative and effective clinical and therapeutic follow-up. The majority of episodes of evaluable pulmonary tuberculosis (49 cases out of 65: 75,4%) occurred in patients who immigrated from developing countries. In two patients multiresistant (MDR) Mycobacterium tuberculosis strains were found, while two more subjects (both immigrated from Eastern Europe) suffered from a disease due to extremely resistant (XDR) M. tuberculosis strains. Although enforcing all possible measures to increase patients' adherence to treatment (empowerment, delivery of oral drugs under direct control, use of i.v. formulation whenever possible), over 72% of evaluable patients had a very slow clinical, microbiological, and imaging ameliorement (1-6 months), with persistance of sputum and/or bronchoalveolar lavage (BAL) fluid positive for M.tuberculosis microscopy and/or culture for over 1-4 months (mean 9.2±3.2 weeks), during an apparently adequate treatment. When excluding patients suffering from XDR and MDR tuberculosis, in four subjects we observed that off-label linezolid adjunct together with at least three drugs with residual activity against tuberculosis, led to a significantly more rapid clinical-radiological improvement and negative microbiological search, with consequent possibility to led to a protected discharge, supported by a sequential, oral therapy. Linezolid was also successfully employed in all the four patients with XDR or MDR pulmonary tuberculosis: among these patients, a definitive or temporarily negativization of respiratory secretions, and consequent discharge, was achieved only after linezolid adjunct. Notwithstanding the maintained microbiological susceptibility of M. tuberculosis strains responsible of the great majority of cases of pulmonary tuberculosis to first-line drugs, an unexpected tendency of patients to have a persistingly positive sputum and/or BAL, and to experience prolonged hospitalization for cure and isolation, has been recognized in the last years. No particularly suggestive radiological imaging seems predictive of a so prolonged course, so that we presently lack of clinical and imaging elements which may be predictive of this slow treatment response. The same is for demographic and epidemiological issues, eventual underlying diseases, and clinical presentation, so that a major problem for health care providers is to distinguish upon admission patients who will be prone to have slow therapeutic response and a related prolonged hospitalization. The novel oxazolidinone linezolid is characterized by an affordable in vitro activity against M. tuberculosis, and an extremely elevated intracellular concentration in respiratory tissues. Worldwide, increasing microbiological, pharmacological, and clinical evidences may recommend the use as linezolid adjunct as an off-label salvage treatment of pulmonary tuberculosis refractory to treatment, although not necessarily determined by resistant (MDR-XDR) M. tuberculosis strains. Randomized clinical trials including initially patients with ascertained chemioresistant tuberculosis, are strongly warranted

Antiangiogenic role of somatostatin receptor 2 in a model of hypoxia-induced neovascularization in the retina: Results from transgenic mice

PURPOSE. To determine whether the somatostatin receptor 2 (sst2) influences angiogenesis and its associated factors in a model of hypoxia-induced retinal neovascularization. METHODS. sst1-knockout (KO) mice, in which sst2 is overexpressed and overfunctional, and sst2-KO mice were used. Angiogenesis was evaluated in fluorescein-perfused retinas. Angiogenesis- associated factors were determined by RT-PCR and immunohistochemistry. RESULTS. Retinal neovascularization was increased in sst2-KO mice, but remained unchanged in sst1-KO compared with wild-type (WT) mice. Retinal levels of sst2 mRNA were not affected by hypoxia. Normoxic levels of angiogenesis regulators were similar in WT and KO retinas except for mRNA levels of IGF-1, Ang-2, and its receptor Tie-2. In WT, hypoxia induced an increase in mRNA levels of (1) VEGF and its receptors, (2) IGF-1R, and (3) Ang-2 and Tie-2. The increase in VEGF and IGF-1R mRNAs was more pronounced after sst2 loss, but was less pronounced when sst2 was overexpressed. In addition, in hypoxic retinas, sst2 loss increased IGF-1 mRNA, whereas it decreased Ang-1, Tie-1, and Tie-2 mRNA levels. Moreover, Tie-1 mRNA increased when sst2 was overexpressed. Immunohistochemistry confirmed the results in hypoxic retinas on increased expression of VEGF, IGF-1, and their receptors after sst2 loss. It also allowed the localization of these factors to specific retinal cells. In this respect, VEGFR-2, IGF-1, and IGF-1R were localized to Mu¨ller cells. CONCLUSIONS. These results suggest that sst2 may be protective against angiogenesis. The immediate clinical importance lies in the establishment of a potential pharmacological target based on sst2 pharmacology.L'articolo è disponibile sul sito dell'editore http://www.arvo.org/eweb/StartPage.aspx?Site=arvo

The β3 adrenoceptor in proliferative retinopathies: "Cinderella" steps out of its family shadow

: In the retina, hypoxic condition leads to overgrowing leaky vessels resulting in altered metabolic supply that may cause impaired visual function. Hypoxia-inducible factor-1 (HIF-1) is a central regulator of the retinal response to hypoxia by activating the transcription of numerous target genes, including vascular endothelium growth factor, which acts as a major player in retinal angiogenesis. In the present review, oxygen urge by the retina and its oxygen sensing systems including HIF-1 are discussed in respect to the role of the beta-adrenergic receptors (β-ARs) and their pharmacologic manipulation in the vascular response to hypoxia. In the β-AR family, β1- and β2-AR have long been attracting attention because their pharmacology is intensely used for human health, while β3-AR, the third and last cloned receptor is no longer increasingly emerging as an attractive target for drug discovery. Here, β3-AR, a main character in several organs including the heart, the adipose tissue and the urinary bladder, but so far a supporting actor in the retina, has been thoroughly examined in respect to its function in retinal response to hypoxia. In particular, its oxygen dependence has been taken as a key indicator of β3-AR involvement in HIF-1-mediated responses to oxygen. Hence, the possibility of β3-AR transcription by HIF-1 has been discussed from early circumstantial evidence to the recent demonstration that β3-AR acts as a novel HIF-1 target gene by playing like a putative intermediary between oxygen levels and retinal vessel proliferation. Thus, targeting β3-AR may implement the therapeutic armamentarium against neovascular pathologies of the eye

Novel Insights into Beta 2 Adrenergic Receptor Function in the rd10 Model of Retinitis Pigmentosa

Background: In retinitis pigmentosa (RP), inherited rod death is followed by cone loss and blindness. Why cones die is still a matter of consideration. Here, we investigate the pathogenic role of the sympathetic transmission in the rd10 mouse model of RP. Methods: Retinal levels of beta adrenergic receptor (BAR) 2 and norepinephrine (NE) were measured. After administration of the BAR1/2 blocker propranolol or the hypoxia-inducible factor (HIF)-1 activator dimethyloxalylglycine (DMOG), retinal levels of HIF-1α, BAR2 or proteins involved in BAR2 desensitization were also measured. In DMOG treated mice, expression and localization of BAR2, inflammatory markers and cone arrestin were determined. Finally, rd10 mice were subjected to electroretinogram (ERG) analysis to assess rod and cone function. Results: In the rd10 retina, BAR2 overexpression and NE accumulation were found, with BAR2 immunoreactivity localized to Müller cells. BAR2 overexpression was likely due to desensitization defects. Upregulated levels of BAR2 were drastically reduced by propranolol that also restored desensitization defects. Due to the low level of HIF-1 consequent to the hyperoxic environment in the rd10 retina, we hypothesized a link between HIF-1 and BAR2. HIF-1α stabilization with DMOG resulted in i. increased HIF-1α accumulation, ii. decreased BAR2 levels, iii. restored desensitization processes, iv. reduced expression of inflammatory markers and v. increased cone survival without improved retinal function. Conclusions: Our results support a pathogenic role of the sympathetic system in RP that might help to understand why rd10 mice show a positive response to BAR blockers

Restored retinal physiology after administration of niacin with citicoline in a mouse model of hypertensive glaucoma

IntroductionMuch interest has been addressed to antioxidant dietary supplements that are known to lower the risk of developing glaucoma or delay its progression. Among them, niacin and citicoline protect retinal ganglion cells (RGCs) from degeneration by targeting mitochondria, though at different levels. A well-established mouse model of RGC degeneration induced by experimental intraocular pressure (IOP) elevation was used to investigate whether a novel combination of niacin/citicoline has better efficacy over each single component in preserving RGC health in response to IOP increase.MethodsOcular hypertension was induced by an intracameral injection of methylcellulose that clogs the trabecular meshwork. Electroretinography and immunohistochemistry were used to evaluate RGC function and density. Oxidative, inflammatory and apoptotic markers were evaluated by Western blot analysis.ResultsThe present results support an optimal efficacy of niacin with citicoline at their best dosage in preventing RGC loss. In fact, about 50% of RGCs were spared from death leading to improved electroretinographic responses to flash and pattern stimulation. Upregulated levels of oxidative stress and inflammatory markers were also consistently reduced by almost 50% after niacin with citicoline thus providing a significant strength to the validity of their combination.ConclusionNiacin combined with citicoline is highly effective in restoring RGC physiology but its therapeutic potential needs to be further explored. In fact, the translation of the present compound to humans is limited by several factors including the mouse modeling, the higher doses of the supplements that are necessary to demonstrate their efficacy over a short follow up period and the scarce knowledge of their transport to the bloodstream and to the eventual target tissues in the eye

Somatostatin receptors differentially affect spontaneous epileptiform activity in mouse hippocampal slices

Somatostatin-14 (SRIF) reduces hippocampal epileptiform activity but the contribution of its specific receptors (sst1-5) is poorly understood. We have focused on sst1 and sst2 role in mediating SRIF modulation of epilepsy using hippocampal slices of wild type (WT) and sst1 or sst2 knock out (KO) mice. Recordings of epileptiform discharge induced by Mg2+-free medium with 4-aminopyridine were performed from the CA3 region before and after the application of SRIF compounds. In WT mice, SRIF and the sst1 agonist CH-275 reduce epilepsy whereas sst1 blockade with its antagonist SRA-880 increases bursting discharge. Activation of sst2 does not affect bursting frequency unless its agonist octreotide is applied with SRA-880, indicating that sst1 masks sst2-mediated modulation of epilepsy. In sst1 KO mice: i. bursting frequency is lower than in WT; ii. SRIF, CH-275 and SRA-880 are ineffective on epilepsy; iii. octreotide is also devoid of effects, whereas blockade of sst2 with the antagonist D-Tyr8 Cyn 154806 increases bursting frequency. In sst2 KO mice, SRIF ligand effects are similar to those in WT. In the whole hippocampus of sst1 KO mice, sst2 mRNA, protein and binding are higher than in WT and RT-PCR of the CA3 subarea confirms an increase of the sst2 messenger. We conclude that sst1 mediates inhibitory actions of SRIF and that interactions between sst1 and sst2 may prevent sst2 modulation of epilepsy. We suggest that, in sst1 KO mice, activation of over-expressed sst2 reduces bursting frequency, indicating that sst2 density represents the rate-limiting factor for sst2-mediated modulation of epilepsy.L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com

Increased efficacy of dietary supplement containing wax ester-rich marine oil and xanthophylls in a mouse model of dry macular degeneration

Age-related macular degeneration (AMD) is nowadays considered among the retinal diseases whose clinical management lacks established treatment approaches, mainly for its atrophic (dry) form. In this respect, the use of dietary patterns enriched in omega-3 and antioxidant xanthophylls has emerged as a promising approach to counteract dry AMD progression although the prophylactic potential of omega-3 of fish origin has been discussed. Whether enriched availability of omega-3 and xanthophylls may increase the effectiveness of diet supplementation in preventing dry AMD remains to be fully established. The present study aims at comparing the efficacy of an existing orally administered formulation based on lutein and fish oil, as a source of omega-3, with a novel formulation providing the combination of lutein and astaxanthin with Calanus oil (COil), which contains omega-3 together with their precursors policosanols. Using a mouse model of dry AMD based on subretinal injection of polyethylene glycol (PEG)-400, we assessed the comparative efficacy of both formulations on PEG-induced major hallmarks including oxidative stress, inflammation, glial reactivity and outer retinal thickness. Dietary supplementation with both mixtures has been found to exert a significant antioxidant and anti-inflammatory activity as reflected by the overall amelioration of the PEG-induced pathological hallmarks. Noteworthy, the formulation based on COil appeared to be more protective than the one based on fish oil, presumably because of the higher bioavailability of omega-3 in COil. These results support the use of dietary supplements combining omega-3 and xanthophylls in the prevention and treatment of AMD and suggest that the source of omega-3 might contribute to treatment efficacy

Somatostatin coupling to adenylyl cyclase activity in the mouse retina

The peptide somatostatin-14 (SRIF) acts in the mammalian retina through its distinct receptors (sst1-5). Scarce information is available on SRIF function in the retina, including the elucidation of transduction pathways mediating SRIF action. We have investigated SRIF and SRIF receptor modulation of adenylyl cyclase (AC) activity in both wild type (WT) retinas and sst1 or sst2 knock-out (KO) retinas which are known to over-express sst2 or sst1 receptors, respectively. In WT retinas, application of SRIF compounds does not affect forskolin-stimulated AC activity. In contrast, activation of sst1 or sst2 receptors inhibits AC in the presence of sst2 or sst1 receptor antagonists, respectively. Results from sst1 KO retinas demonstrate that either SRIF or octreotide, pertussis toxin-dependently inhibit AC activity. In contrast, in sst2 KO retinas, neither SRIF nor CH-275, an sst1 receptor agonist, are found to influence AC activity. As revealed by immunoblotting experiments, in sst1 KO retinas, levels of Goα proteins are 60% higher than in WT retinas and this increase in Goα protein levels is concomitant with an increase in sst2A receptor expression. We conclude that interactions between sst1 and sst2 receptors may prevent SRIF effects on AC activity. In addition, we suggest that the density of sst2 receptors and/or Goα proteins may represent the rate-limiting factor for the sst2 receptor-mediated inhibition of AC.L'articolo è disponibile sul sito dell'editore http://www.springerlink.com

A Topical Formulation of Melatoninergic Compounds Exerts Strong Hypotensive and Neuroprotective Effects in a Rat Model of Hypertensive Glaucoma

Melatonin is of great importance for regulating several eye processes, including pressure homeostasis. Melatonin in combination with agomelatine has been recently reported to reduce intraocular pressure (IOP) with higher efficacy than each compound alone. Here, we used the methylcellulose (MCE) rat model of hypertensive glaucoma, an optic neuropathy characterized by the apoptotic death of retinal ganglion cells (RGCs), to evaluate the hypotensive and neuroprotective efficacy of an eye drop nanomicellar formulation containing melatonin/agomelatine. Eye tissue distribution of melatonin/agomelatine in healthy rats was evaluated by HPLC/MS/MS. In the MCE model, we assessed by tonometry the hypotensive efficacy of melatonin/agomelatine. Neuroprotection was revealed by electroretinography; by levels of inflammatory and apoptotic markers; and by RGC density. The effects of melatonin/agomelatine were compared with those of timolol (a beta blocker with prevalent hypotensive activity) or brimonidine (an alpha 2 adrenergic agonist with potential neuroprotective efficacy), two drugs commonly used to treat glaucoma. Both melatonin and agomelatine penetrate the posterior segment of the eye. In the MCE model, IOP elevation was drastically reduced by melatonin/agomelatine with higher efficacy than that of timolol or brimonidine. Concomitantly, gliosis-related inflammation and the Bax-associated apoptosis were partially prevented, thus leading to RGC survival and recovered retinal dysfunction. We suggest that topical melatoninergic compounds might be beneficial for ocular health

Hypotensive Effect of Nanomicellar Formulation of Melatonin and Agomelatine in a Rat Model: Significance for Glaucoma Therapy

Background: Melatoninergic agents are known to reduce intraocular pressure (IOP). The present study was performed to evaluate the effect of nanomicellar formulations of melatoninergic agents on IOP in the rat. Methods: Tonometry was used to measure IOP in eyes instilled with melatonin or agomelatine. Ocular hypertension was induced by the injection of methylcellulose in the anterior chamber. Results: Melatonin formulated in nanomicelles had a longer lasting hypotonizing effect on IOP with respect to melatonin in saline. Nanomicellar formulations of melatonin and agomelatine, either alone or in combination, had lowering effects that did not depend on their concentration or their combination, which, however, resulted in an increased duration of the hypotonizing effect. The duration of the lowering effect was further increased by the addition of lipoic acid. Conclusions: We demonstrated the effective hypotonizing activity of melatonin and agomelatine in combination with lipoic acid. Although results in animals cannot be directly translated to humans, the possibility of developing novel therapeutical approaches for patients suffering from hypertensive glaucoma should be considered