Comment on: "A case report of a complete degloving injury of the penile skin".

INTRODUCTION: We recently published an article in the International Journal of Surgical Case Reports titled: "Scrotal dartos-fascio-myo-cutaneous flap for penis elongation after catastrophic iatrogenic skin shaft sub-amputation: A case of recovery using an extremely adaptable flap". PRESENTATION OF CASE: We propose a comment on a recent article titled "A case report of a complete degloving injury of the penile skin" by Helena Aineskog and Frederik Huss that we read with great interest. DISCUSSION: Genitalia are linked to self-esteem and male sexual identity, especially among young men, who sometimes require a surgical procedure to acquire more confidence. Various techniques are available for pe-nile skin covering, such as skin grafts or cutaneous flaps. The skin of the scrotum seems to be the most suitable tissue to be used to reconstruct the skin covering of the shaft as it is the most similar. CONCLUSION: Scrotal flap is a single stage procedure that is easy and safe to perform.

Tumor MGMT promoter hypermethylation changes over time limit temozolomide efficacy in a phase II trial for metastatic colorectal cancer.

BACKGROUND: Objective response to dacarbazine, the intravenous form of temozolomide (TMZ), in metastatic colorectal cancer (mCRC) is confined to tumors harboring O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter hypermethylation. We conducted a phase II study of TMZ enriched by MGMT hypermethylation in archival tumor (AT), exploring dynamic of this biomarker in baseline tumor (BT) biopsy and plasma (liquid biopsy). PATIENTS AND METHODS: We screened 150 mCRC patients for MGMT hypermethylation with methylation-specific PCR on AT from FFPE specimens. Eligible patients (n = 29) underwent BT biopsy and then received TMZ 200 mg/m(2) days 1-5 q28 until progression. A Fleming single-stage design was used to determine whether progression-free survival (PFS) rate at 12 weeks would be ≥35% [H0 ≤ 15%, type I error = 0.059 (one-sided), power = 0.849]. Exploratory analyses included comparison between MGMT hypermethylation in AT and BT, and MGMT methylation testing by MethylBEAMing in solid (AT, BT) and LB with regard to tumor response. RESULTS: The PFS rate at 12 weeks was 10.3% [90% confidence interval (CI) 2.9-24.6]. Objective response rate was 3.4% (90% CI 0.2-15.3), disease control rate 48.3% (90% CI 32.0-64.8), median OS 6.2 months (95% CI 3.8-7.6), and median PFS 2.6 months (95% CI 1.4-2.7). We observed the absence of MGMT hypermethylation in BT in 62.7% of tumors. CONCLUSION: Treatment of mCRC with TMZ driven by MGMT promoter hypermethylation in AT samples did not provide meaningful PFS rate at 12 weeks. This biomarker changed from AT to BT, indicating that testing BT biopsy or plasma is needed for refined target selection.

Different patterns characterize Omega 6 and Omega 3 long chain polyunsaturated fatty acid levels in blood from Italian infants, children, adults and elderly.

Long chain polyunsaturated fatty acids (LC-PUFA), especially the Omega 3, modulate key functions in the body. Their circulating levels are representative of their "status", and may vary at different ages. We have compared the FA status in Italian subjects from neonates to adulthood, assessed through FA analysis of blood drops from fingertips. Data from four cohorts of Italian subjects (total number 1835), have been pooled in four age-groups: neonates (4 days, n=81), children (2-9 years, n=728), adults (40-59 years, n=434) and elderly (60-79 years, n=592). LC-PUFA of both series (Omega 3 and 6) are higher in the blood of neonates than at subsequent ages, reflecting the efficient transfer of these FA from mothers to the fetus. In contrast, the lowest levels of Omega 3 PUFA, especially of DHA, are found in children, probably reflecting inadequate dietary intakes, with possible consequences on the health status at subsequent ages.

Persistence of CCR5 usage among primary human immunodeficiency virus isolates of individuals receiving intermittent interleukin-2.

OBJECTIVE: To investigate the impact of intermittent interleukin-2 (IL-2) plus combination antiretroviral therapy (cART) on HIV-1 entry co-receptor use. METHODS: Primary HIV-1 isolates were obtained from 54 HIV-1-positive individuals at baseline and after 12 months using co-cultivation of peripheral blood mononuclear cells (PBMC) with activated PBMC of HIV-negative healthy donors. HIV-1 co-receptor use was determined on U87-CD4 cells. RESULTS: Fourteen out of the 21 (67%) IL-2-treated individuals harbouring a primary CCR5-dependent (R5) HIV-1 isolate at baseline confirmed an R5 virus isolation after 12 months in contrast to 3 out of 7 (43%) of those receiving cART only. After 12 months, only 1 R5X4 HIV-1 isolate was obtained from 21 cART+IL-2-treated individuals infected with an R5 virus at entry (5%) vs. 2/7 (29%) patients receiving cART alone, as confirmed by a 5-year follow-up on some individuals. CONCLUSIONS: Intermittent IL-2 administration plus cART may prevent evolution towards CXCR4 usage in individuals infected with R5 HIV-1.

TARDBP (TDP-43) sequence analysis in patients with familial and sporadic ALS: identification of two novel mutations.

BACKGROUND AND PURPOSE: Increasing evidence suggests a direct role of the TAR DNA-binding protein 43 (TDP-43) in neurodegeneration. Mutations in the TARDBP gene, which codes for TDP-43, have been recently reported in familial and sporadic amyotrophic lateral sclerosis (ALS) cases. METHODS: To further define the spectrum and frequency of TARDBP mutations, we present genetic analysis data on TARDBP in 314 ALS mainly Italian patients, including 16 subjects with non-SOD1 familial ALS. RESULTS: We identified four heterozygous missense mutations in five unrelated ALS patients (1.6%). Two of these mutations (p.G348C and p.A382T) were detected in carriers coming from families with an autosomal dominant transmission of different geographic origin (Belgian and Italian, respectively). The Belgian pedigree showed several affected members within five generations and with variable clinical features. Two novel mutations (p.G294V and p.G295S) were identified in two sporadic cases. CONCLUSION: The identification of five ALS patients carrying TARDBP alterations extends the spectrum of TARDBP mutations and supports the pathological role of TDP-43 in motor neurone disease. Our findings provide evidence that TARDBP mutations are not frequent in Italian sporadic ALS patients (1%); however, combined with the literature, our data further support TARDBP mutations as a relevant cause of familial ALS.

The in vitro effects of cigarette smoke on fatty acid metabolism are partially counteracted by simvastatin.

BACKGROUND: Statins enhance the synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) from their precursors both in vitro and in vivo. In particular, an increased conversion of linoleic acid (LA) and of alpha-linolenic acid to their derivatives is observed in cultured cells. On the contrary, cigarette smoke (CS) negatively and dose-dependently affects the LC-PUFA production. AIM: To evaluate the effects of CS alone or with simvastatin, on [1-(14)C] LA metabolism in THP-1 cells. RESULTS: CS inhibits LA conversion; after co-incubation, simvastatin nullifies the effects of CS, maintaining LA conversion comparable to controls. However, at the highest CS concentration, simvastatin is unable to counteract the effects of CS. Changes of LA conversion reflect the modulation of desaturase activities by simvastatin and CS. CONCLUSION: CS decreases PUFA conversion and its effects are modulated by the opposite effect of statins. It can be speculated that statin treatments in smoking patients may provide some beneficial effects on PUFA metabolism in addition to lowering cholesterol levels.

Mutated mitofusin 2 presents with intrafamilial variability and brain mitochondrial dysfunction.

BACKGROUND: The axonal forms of Charcot-Marie-Tooth (CMT2) disease are a clinically and genetically heterogeneous group of disorders. Mitofusin 2 gene (MFN2) mutations are the most common cause of CMT2. Complex phenotypes have been described in association with MFN2 gene mutations, including CMT2 with pyramidal features (hereditary motor and sensory neuropathy [HSMN V]) and CMT2 with optic atrophy (HMSN VI). OBJECTIVE: To report on the clinical, neurophysiologic, and neuropathologic features of an Italian family with a novel MFN2 gene mutation and investigate brain functional parameters using magnetic resonance spectroscopy (MRS). METHODS: Three family members, a father and his two sons, were affected by peripheral neuropathy, cognitive impairment, and poor nocturnal vision (also optic neuropathy in one case). A member of this family also showed spastic paraparesis. The MFN2 gene sequence was analyzed. A sural nerve biopsy as well as brain (1)H-MRS and (31)P-MRS were evaluated in two patients. RESULTS: Affected family members carried a novel MFN2 missense mutation, namely R104W, located within the critical GTPase domain of the protein which affects a highly conserved amino acid position. Sural nerve biopsies showed a normal mitochondrial network, particularly at the nodes of Ranvier, upon electron microscopy examination. A significant defect of high energy phosphates (HEPs) in the visual cortex was observed at rest by (31)P-MRS in the adult proband, while his son showed a defective recovery of HEPs after stimulation of the visual cortex. CONCLUSION: Cognitive impairment may be another feature of the MFN2-related phenotype. The widespread peripheral and CNS involvement, as well as the neurosensorial defects, underline the similarities among MFN2-related and primary mitochondrial disorders.

Preliminary evidence that VEGF genetic variability confers susceptibility to frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) recognizes a strong genetic background, with 30-50% of cases with a positive family history. Despite several efforts to identify monogenic causes of the disease, no clear-cut genetic risk factors for sporadic FTLD are yet known. Recently, increasing evidence points to a pivotal role of vascular endothelial growth factor (VEGF) in the neurodegenerative process, suggesting functions not confined to its originally described vascular effects. The aim of this study was to investigate the role of VEGF as a genetic determinant to FTLD susceptibility. We evaluated a cohort of 274 unrelated Italian patients, including 161 subjects with frontotemporal dementia (FTD), 56 with corticobasal degeneration syndrome, and 57 with progressive supranuclear palsy. Genotype and allele frequencies of four well-known polymorphisms located within the VEGF promoter (-2578C/A, -1190G/A, -1154G/A, and -634G/C) were calculated in patients and in 216 age-matched healthy subjects. Genetic analysis revealed the presence of several significant changes in terms of allele, genotype, and haplotype frequency distributions between patients and controls. Marked differences were observed when the FTD patient subgroup was compared with healthy subjects. Overall, these data provide evidence for the first time that VEGF gene variability represents a susceptibility factor for sporadic FTLD, at least in an Italian population. Future confirmatory studies are mandatory.

Fatty acid composition of plasma, blood cells and whole blood: relevance for the assessment of the fatty acid status in humans.

UNLABELLED: The composition and incorporation of fatty acids (FA) in plasma and blood cells is the result of distinct processes: intake, metabolism and peripheral utilization. AIM OF THE STUDY: was to compare the FA profile in plasma, lipoproteins and blood cells with that in whole blood (WB) from healthy volunteers; to assess the quantitative distribution of selected FA in triacylglycerols, cholesteryl esters and phospholipids. Lipid FA profiles are comparable in plasma and lipoproteins but differ from those in blood cells. In WB, the FA profile results from the balanced proportion of FA pools in plasma and cells. The contribution of each lipid class to the total amount of FA differs among blood specimens. Phospholipids of plasma and red blood cell are the major contributors to the FA amount and profile in WB. In conclusion, the FA profile of WB reflects the FA status and WB could be an adequate specimen for the assessment of FA intakes.

Coexistence of CMT-2D and distal SMA-V phenotypes in an Italian family with a GARS gene mutation.

An Italian multigenerational family with four members affected by an axonal Charcot-Marie-Tooth type 2D (CMT-2D) or distal spinal muscular atrophy (dSMA) phenotype with upper limb predominance, variable age at onset, degree of disability, and autosomal dominant inheritance is reported. A novel heterozygous missense GARS gene mutation (D500N) was identified.

Synthesis of long-chain polyunsaturated fatty acids is inhibited in vivo in hypercholesterolemic rabbits and in vitro by oxysterols.

Plasma total lipids, total cholesterol (cholesterol esters and free cholesterol) and oxysterol (mainly 7 beta-hydroxycholesterol (7 beta OH)) concentrations were significantly elevated in New Zealand rabbits fed a 2% cholesterol-containing diet with respect to controls fed the same diet without cholesterol. In addition, linoleic (18:2 n-6) and alpha-linolenic acid (18:3 n-3) plasma concentrations were significantly elevated in hypercholesterolemic rabbits, while concentrations of long-chain n-6 and n-3 derivatives were reduced. Studies in monocytic cell line THP-1 revealed that 7 beta OH markedly inhibited the conversion of 18:2 to 20:4 n-6 and of 18:3 to 22:6 n-3, indicating depression of the desaturation steps; in particular the inhibition was greater for the Delta 5 desaturation step. Furthermore, experiments of Real-Time PCR showed that 5-10 microM 7 beta OH decreased the Delta 5 gene expression. In conclusion, atherogenic oxysterols interfere with the production of long-chain polyunsaturated fatty acids from their precursors both in hypercholesterolemic rabbits and in cultured cells.

Sulfated K5 Escherichia coli polysaccharide derivatives inhibit human immunodeficiency type-1 (HIV-1) infection: candidate microbicides to prevent sexual HIV transmission.

The ideal microbicide must fulfill a number of criteria including a broad and potent activity against transmission of HIV and other sexually transmitted agents in the absence of toxicity and inflammation. We have described that derivatives of K5 polysaccharide from Escherichia coli inhibit HIV entry in target cells. K5 derivatives have a structure that resembles that of heparin, but they are devoid of the anticoagulant activity typical of heparin. Moreover, in contrast to heparin, they inhibit a broad spectrum of HIV-1 laboratory-adapted and primary isolates that use either CCR5 or CXCR4 or both coreceptors in terms of their infection and replication in primary CD4+ lymphocytes and monocytes-derived macrophages (MDM). Therefore, these compounds could be developed as candidate microbicides for preventing sexual HIV transmission, a predominant modality of HIV spreading in both the developed and underdeveloped world.

Pharmacological modulation of fatty acid desaturation and of cholesterol biosynthesis in THP-1 cells.

In THP-1 cells, simvastatin decreases, in a concentration-dependent manner, cholesterol synthesis and increases linoleic acid (LA) conversion to its long-chain derivatives, in particular to arachidonic acid, activating delta6 and delta5 fatty acid (FA) desaturases. The intermediates in cholesterol synthesis, mevalonate and geranylgeraniol, partially reverse the effects of simvastatin on the LA conversion. The aims of this work were to evaluate: (i) the correlation between cholesterol synthesis and desaturase activity and (ii) the possible involvement of protein isoprenylation in desaturase activity, assessed through pharmacological treatments. THP-1 cells were incubated with [1-14C]LA or with [1-14C]di-homo-gamma-linolenic acid (DHGLA) and treated with simvastatin or with curcumin and nicardipine, inhibitors of desaturases. Curcumin was more active than nicardipine in inhibiting LA and DHGLA conversion: 20 microM curcumin, alone or with simvastatin, totally inhibited delta6 and delta5 desaturation steps; 10 microM nicardipine only partially inhibited the enzymes, being more active on delta5 desaturase. Simvastatin treatment decreased the incorporation of acetate in cholesterol (-93.8%) and cholesterol esters (-70.2%), as expected. Curcumin and nicardipine also decreased cholesterol synthesis and potentiated simvastatin. Finally, the isoprenylation inhibitors (perillic acid and GGTI-286) neither affected the conversion of LA nor inhibited the delta5 desaturase activity. In conclusion, our results indicate that there is no direct relationship between cholesterol synthesis and desaturase activity. In fact, simvastatin decreased cholesterol synthesis and enhanced LA conversion (mainly delta5 desaturation), whereas curcumin and nicardipin decreased delta5 desaturation, with a limited effect on cholesterol synthesis.

Spreading of HIV-specific CD8+ T-cell repertoire in long-term nonprogressors and its role in the control of viral load and disease activity.

Long-term non-progressors (LTNP) represent a minority of human immunodeficiency virus (HIV) infected individuals characterized by stable or even increasing CD4+ T-cell count and by stronger immune responses against HIV than progressors. In this study, HIV-specific effector CD8+ T cells, as detected by both a sensitive ex vivo enzyme-linked immunospot (ELISPOT) assay and specific major histocompatibility complex (MHC) peptide tetramers, were at a low frequency in the peripheral blood of LTNP, and recognized a lower number of HIV peptides than their memory resting cell counterparts. Both factors may account for the lack of complete HIV clearance by LTNP, who could control the viral spread, and displayed a higher magnitude of cytotoxic T lymphocyte (CTL) responses than progressors. By combining cell purification and ELISPOT assays this study demonstrates that both effector and memory resting cells were confined to a CD8+ population with memory CD45RO+ phenotype, with the former being CD28- and the latter CD28+. Longitudinal studies highlighted a relatively stable HIV-specific effector repertoire, viremia, and CD4+ T-cell counts, which were all correlated with maintenance of nonprogressor status. In conclusion, the analysis of HIV-specific cellular responses in these individuals may help define clear correlates of protective immunity in HIV infection.

Expression and activation of a C-terminal truncated isoform of STAT5 (STAT5 Delta) following interleukin 2 administration or AZT monotherapy in HIV-infected individuals.

Intermittent administration of recombinant interleukin-2 (rIL-2) to individuals infected with human immunodeficiency virus (HIV) has been shown to raise and maintain the absolute number of circulating CD4(+) T cells to normal or near normal levels. One of the signaling pathways triggered by IL-2 is the Janus kinase-signal transducer and activator of transcription (JAK-STAT). In particular, IL-2 activates the tyrosine kinases JAK1 and JAK3 and the transcription factors STAT3 and STAT5. We have previously observed that most HIV(+) individuals, unlike healthy seronegative controls, show a constitutive activation of STAT1 and a C-terminal truncated isoform of STAT5 (STAT5 Delta). In the present study, we have analyzed the protein level and activation state of STAT5 isoforms expressed in peripheral blood mononuclear cells of two HIV-infected individuals who showed a good or a poor response to intermittent IL-2 administration, respectively, and of a single individual before and after initiation of Zidovudine monotherapy. We provide evidence that both therapeutic interventions enhanced the expression and activation of the C-terminal truncated isoform of STAT5 (STAT5 Delta) in vivo.

Efficacy of low-dose intermittent subcutaneous interleukin (IL)--2 in antiviral drug--experienced human immunodeficiency virus--infected persons with detectable virus load: a controlled study of 3 il-2 regimens with antiviral drug therapy.

To evaluate the safety and efficacy of 3 regimens of intermittent subcutaneous (sc) interleukin (IL)--2 in a phase 2 study, 61 antiviral drug-experienced human immunodeficiency virus (HIV)--positive patients were randomly assigned to one of the following study arms: antiretroviral therapy (ART) plus IL-2 (12 million IU [MIU] by continuous intravenous infusion, followed by 7.5 MIU twice a day, sc, every 8 weeks); ART plus IL-2 (7.5 MIU twice a day, sc, every 8 weeks); ART plus IL-2 (3 MIU twice a day, sc, every 4 weeks); or ART alone. A significant increase of circulating CD4 cells was observed in IL-2--treated subjects, compared with those given ART alone. Low doses of IL-2 were better tolerated. Despite the incomplete suppression of viral replication, IL-2 with ART did not increase either plasma viremia or cell-associated HIV DNA levels. Low doses of intermittent sc IL-2 induced a stable increase of peripheral CD4 cells that was indistinguishable from those associated with higher, less well-tolerated doses of IL-2.

Inhibition of R5X4 dualtropic HIV-1 primary isolates by single chemokine co-receptor ligands.

The susceptibility of HIV-1 to chemokine-mediated inhibition may be lost as a consequence of the expanded usage of chemokine co-receptors frequently occurring in clade B isolates obtained from individuals with advanced disease. Since chemokine-based immune intervention is under intense investigation, it is crucial to determine its potential effect on primary dualtropic HIV isolates characterized by simultaneous utilization of CCR5 and CXCR4 chemokine co-receptors (R5X4 viruses). In the present study, the CCR5 binding chemokine regulated upon activation normal T cell expressed and secreted (RANTES) strongly inhibited the replication of two of eight primary R5X4 viruses in mitogen-activated primary peripheral blood mononuclear cells (PBMC). The CXCR4 antagonist AMD3100 efficiently suppressed the replication of other two HIV isolates, whereas the remaining four viruses were partially inhibited by treatment with either RANTES or AMD3100. The potency of chemokine-mediated inhibition was influenced by PBMC donor variability, but it was usually independent from the levels of expression of CCR5 or CXCR4. Dual co-receptor usage was maintained by the viruses after two serial passages on U87.CD4 astrocytic cell lines expressing exclusively either CCR5 or CXCR4. The gp120 env variable domains were sequenced before and after passages on U87.CD4 cells. Virus replication into U87.CD4-CXCR4 cells did not result in changes in the V3 region but perturbed the dominant env V4 sequence. Interestingly, double passage onto U87.CD4-CXCR4 cells determined the loss of susceptibility to RANTES inhibition. In conclusion, interference with CCR5 may efficiently inhibit the replication of at least some dualtropic HIV-1 strains, whereas forced CXCR4 usage may result in viral escape from CCR5-dependent inhibitory effects.

Divergent regulation of HIV-1 replication in PBMC of infected individuals by CC chemokines: suppression by RANTES, MIP-1alpha, and MCP-3, and enhancement by MCP-1.

We investigated the role of different CC chemokines, including regulated upon activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-lalpha (MIP-1alpha), monocyte chemotactic protein-1 (MCP-1), and MCP-3 on virus replication in cultures established from CD8+ T cell-depleted peripheral blood mononuclear cells (PBMC) of HIV-infected individuals that were either cocultivated with allogeneic T cell blasts (ATCB) of uninfected individuals or directly stimulated by mitogen plus interleukin-2. RANTES was the only chemokine that showed a clear-cut suppressive effect on HIV replication in both culture systems, although inhibitory effects were frequently also observed with MIP-1alpha, MCP-3, and, occasionally, with MCP-1. In contrast, MCP-1 frequently enhanced HIV production in most patients' cultures or cocultures that were characterized by secreting relatively low levels (<20 ng/mL) of MCP-1. When CD8-depleted PBMC of HIV+ individuals were cocultivated with ATCB of uninfected healthy donors, a positive correlation was observed between MCP-1 concentrations and the enhancement of HIV-1 replication occurring after depletion of CD8+ cells from donors' cells. Depletion of CD14+ cells (monocytes) from ATCB resulted in the down-regulation of virus replication during co-cultivation with CD8-depleted PBMC of infected individuals. Of interest, MCP-1 up-regulated HIV production in these CD14-depleted ATCB cocultures. Altogether these observations suggest that MCP-1 may represent an important factor enhancing HIV spreading, particularly in anatomical sites, such as the brain, where infection of macrophages and microglial cells plays a dominant role.