Natural history of a cohort of ABCD1 variant female carriers.

BACKGROUND AND PURPOSE: The therapeutic scenario of X-linked adrenoleukodystrophy (X-ALD) is rapidly changing. Whereas the disease is well characterized in men, the condition remains to be fully clarified in women carrying ATP binding cassette subfamily D member 1 (ABCD1) variants. Specifically, data on clinical progression are needed, in order to recommend any appropriate management. The objective of this study was to outline the natural history of a cohort of untreated ABCD1 heterozygous female carriers. METHODS: Longitudinal data from a single-center population of 60 carriers were retrospectively reviewed. Demographics, anthropometrics, serum very long chain fatty acid (VLCFA) levels, clinical parameters and the Adult ALD Clinical Score (AACS) were collected from every recorded visit in a 7-year period and analyzed to define the phenotype modifications, to determine factors associated with clinical features, and to estimate the annual progression rate and the subsequent sample size for interventional trials. RESULTS: Thirty-two patients were eligible for the study, and 59.4% were symptomatic at baseline. Clinical severity worsens with age which increases risk of symptom onset, the cut-off of 41 years being crucial for phenoconversion. VLCFA levels were not predictive and did not change over time. Symptomatic carriers were followed up for 3.45 ± 2.1 years. The AACS increased at an annual rate of 0.24 points. The estimated sample size for 30% reduction in annual progression at 80% power was 272. CONCLUSIONS: This study provides data on the natural disease progression of untreated ABCD1 heterozygous female carriers, demonstrating the relevance of aging. The estimated annual increase of the AACS will be useful for future interventional studies.

The slow dynamics of mycoplasma infections in a tortoise host reveal heterogeneity pertinent to pathogen transmission and monitoring.

The epidemiology of infectious diseases depends on many characteristics of disease progression, as well as the consistency of these processes across hosts. Longitudinal studies of infection can thus inform disease monitoring and management, but can be challenging in wildlife, particularly for long-lived hosts and persistent infections. Numerous tortoise species of conservation concern can be infected by pathogenic mycoplasmas that cause a chronic upper respiratory tract disease (URTD). Yet, a lack of detailed data describing tortoise responses to mycoplasma infections obscures our understanding of URTDs role in host ecology. We therefore monitored Mycoplasma agassizii infections in 14 captive desert tortoises and characterised clinical signs of disease, infection intensity, pathogen shedding and antibody production for nearly 4 years after initial exposure to donor hosts. Persistent infections established in all exposed tortoises within 10 weeks, but hosts appeared to vary in resistance, which affected the patterns of pathogen shedding and apparent disease. Delays in host immune response and changes to clinical signs and infection intensity over time resulted in inconsistencies between diagnostic tools and changes in diagnostic accuracy throughout the study. We discuss the implications these results have for URTD epidemiology and past and future research assessing disease prevalence and dynamics in tortoise populations.

Intensive lifestyle intervention is particularly advantageous in poorly controlled type 2 diabetes.

BACKGROUND AND AIMS: It is unknown whether lifestyle change is effective in people with type 2 diabetes with inadequate glucose control. The aim of this study was to asses, in a group of people with type 2 diabetes, the impact of baseline values of glycosylated haemoglobin (HbA1c) on the effects of an intensive lifestyle intervention on metabolic, clinical and strength parameters. METHODS AND RESULTS: 222 people with type 2 diabetes with mean ± standard deviation baseline HBA1c of 7.50% ± 1.27 (range 5.1-12.7%), were enrolled in a 3-month structured multidisciplinary lifestyle intervention. Anthropometric, biochemical, clinical and fitness measurements were collected at baseline, at the end of the lifestyle intervention program and at two-year follow-up visit. Significant improvements in glycometabolic control (HbA1c: p ≤ 0.0001); anthropometric parameters (BMI p ≤ 0.0001; waist circumference: p ≤ 0.0001); and systemic blood pressure (p ≤ 0.0001) were observed both at the end of the three month intensive lifestyle program and at the two-year follow up visit. In addition, defined daily doses of hypoglycaemic treatment significantly decreased (p = 0.001). Fitness measures exhibited significant increments in the whole sample at the end of the intensive intervention program (p ≤ 0.0001). When patients were divided into tertiles considering the baseline value of HbA1c, the most marked improvements in HbA1c, blood glucose and triglycerides were observed in the group with inadequate glucose control (Hba1c ≥ 7.71%), both at the three-month and two-year follow-ups. CONCLUSION: These results demonstrate that an intensive lifestyle intervention should be recommended for people with type 2 diabetes, particularly those with the most inadequate glycaemic control. REGISTRATION NUMBER: CURIAMO trial was registered in the Australian New Zealand Clinical Trials Registry, (ACTRN12611000255987).

High-resolution DNA content analysis of microbiopsy samples in oral lichen planus.

OBJECTIVES: DNA aneuploidy has been reported to be a predictor of poor prognosis in both premalignant and malignant lesions. In oral lichen planus (OLP), this hypothesis remains to be proved. This study aimed to determine the rate of occurrence of DNA aneuploidy in patients with OLP by high-resolution DNA flow cytometry. METHODS: Patients with OLP were consecutively enrolled. Tissue samples were subdivided for formalin fixation and routine histological assessment and for immediate storage at -20°C for later DNA ploidy analysis, which was performed by DAPI staining of the extracted nuclei and excitation with a UV lamp. The DNA aneuploid sublines were characterized by the DNA Index. RESULTS: A DNA aneuploid status was observed in two of 77 patients with OLP (2.6%). When considering the clinical aspect of the OLP lesions, both DNA aneuploid cases had a reticular clinical aspect. CONCLUSIONS: DNA aneuploidy is an uncommon event in OLP and less frequent compared to other non-dysplastic and non-OLP oral potentially malignant disorders. The extremely low rate of DNA aneuploidy could represent an occasional finding or reflect the low rate of malignant transformation observed in patients with OLP even if the real prognostic value of DNA ploidy analysis in patients with OLP remains to be confirmed.

Is subclinical adrenal failure in adrenoleukodystrophy/adrenomyeloneuropathy reversible?

BACKGROUND: X-linked adrenoleukodystrophy/adrenomieloneuropathy (ALD/AMN) is a progressive neurodegenerative disorder due to mutations in the ABCD1 gene encoding the ABC transporter ALDP. Mutations in ALDP impair peroxisomal ß-oxidation of very long chain fatty acids (VLCFA), resulting in elevated levels of VLCFA in plasma, nervous system, and adrenals. Lorenzo's oil, combined with VLCFA- poor diet, normalizes plasma VLCFA within 1 month, but it does not prevent the progression of pre-existing neurological symptoms. No previous study analyzed the effect of Lorenzo's oil therapy on adrenal function. AIM: To investigate short-term effects of Lorenzo's oil, combined with VLCFA- poor diet, on adrenal function of AMN patients with early subclinical signs of adrenal failure. SUBJECTS AND METHODS: Seven AMN subjects underwent VLCFA-restricted diet combined with Lorenzo's oil (45 ml/day po), without steroid therapy, for 6 months. RESULTS: All patients had elevated ACTH at baseline, and a significant reduction was evident after 6 months (median ACTH at baseline: 1300 pg/ml, range: 720- 2100; median ACTH at 6 months: 186 pg/ml, range: 109-320, p: 0.0156). Cortisol was normal both at baseline and after 6 months. VLCFA dropped in all patients during the 6- month follow-up, and no patient required glucocorticoid replacement therapy. CONCLUSIONS: Adrenal insufficiency in ALD/AMN is probably due to a defective adrenal response to ACTH, related to VLCFA accumulation with progressive disruption of the adrenal cell membrane functions. In an early phase, Lorenzo's oil therapy may be able to improve VLCFA clearance and restore a normal ACTH receptor activity, and hypoadrenalism may be potentially reversible.

Congenital muscular dystrophies with defective glycosylation of dystroglycan: a population study.

BACKGROUND: Congenital muscular dystrophies (CMD) with reduced glycosylation of alpha-dystroglycan (alpha-DG) are a heterogeneous group of conditions associated with mutations in six genes encoding proven or putative glycosyltransferases. OBJECTIVES: The aim of the study was to establish the prevalence of mutations in the six genes in the Italian population and the spectrum of clinical and brain MRI findings. METHODS: As part of a multicentric study involving all the tertiary neuromuscular centers in Italy, FKRP, POMT1, POMT2, POMGnT1, fukutin, and LARGE were screened in 81 patients with CMD and alpha-DG reduction on muscle biopsy (n = 76) or with a phenotype suggestive of alpha-dystroglycanopathy but in whom a muscle biopsy was not available for alpha-DG immunostaining (n = 5). RESULTS: Homozygous and compound heterozygous mutations were detected in a total of 43/81 patients (53%), and included seven novel variants. Mutations in POMT1 were the most prevalent in our cohort (21%), followed by POMT2 (11%), POMGnT1 (10%), and FKRP (9%). One patient carried two heterozygous mutations in fukutin and one case harbored a new homozygous variant in LARGE. No clear-cut genotype-phenotype correlation could be observed with each gene, resulting in a wide spectrum of clinical phenotypes. The more severe phenotypes, however, appeared to be consistently associated with mutations predicted to result in a severe disruption of the respective genes. CONCLUSIONS: Our data broaden the clinical spectrum associated with mutations in glycosyltransferases and provide data on their prevalence in the Italian population.

POMT1 and POMT2 mutations in CMD patients: a multicentric Italian study.

Mutations in POMT1 and POMT2 genes were originally identified in Walker-Warburg syndrome (WWS) and subsequently reported in patients with milder phenotypes characterised by mental retardation with or without brain abnormalities and without ocular malformations. As part of a multicentric Italian study we screened the POMT1 and POMT2 genes in 61 congenital muscular dystrophy (CMD) patients with alpha-dystroglycan reduction on muscle biopsy and/or clinical and radiological findings suggestive of the known forms of CMD with alpha-dystroglycan deficiency. The aim of the study was to establish how frequently mutations in POMT1 and POMT2 occur in CMD patients in the Italian population and to evaluate the spectrum of associated phenotypes. Thirteen patients showed mutations in POMT1 and five harboured mutations in POMT2, accounting for a total of 20 different mutations, eight of which were novel (two in POMT1 and six in POMT2). Normal brain MRI associated with mental retardation and microcephaly was the most frequent finding in patients with mutations in POMT1 (six out of 13), but was also found in a patient with POMT2 mutations. Predominant cerebellar hypoplasia was also frequent both in patients with POMT1 (three out of 13) and POMT2 (three out of 5) mutations. A MEB phenotype with frontal cortical dysplasia and pons abnormalities was found in two patients with POMT1 and in one with POMT2 mutations, while a WWS phenotype was only found in a case with mutations in POMT1. Mutations causing frameshifts and stop codons were responsible for the more severe phenotypes. Our results provide further evidence that, as previously reported for FKRP, the array of mutations in POMT1 and POMT2 is ample and the spectrum of associated phenotypes is wider than initially thought.

A histochemical approach to the evaluation of the in vivo cytotoxicity of the nitrobutadienes (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene and methyl (2Z,4E)-2-methylsulfanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate in mice liver and kidney.

Two new molecules (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1-Naph-DNB) and (2Z,4E)-2-methylsulfanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate (1-Naph-NMCB) in previous studies showed interesting antiproliferative activity in vitro. Furthermore, toxicological tests and histological analysis provided promising results, in particular for 1-Naph-NMCB that displayed lower toxic activity both in terms of lethal effect and tissue damage of the main organs. Finally, studies of the antitumour activity in vivo confirmed the efficacy of both molecules, though with some differences in tumour selectivity and levels of activity. In this investigation the activities of some specific enzymes, acid phosphatase (AcPase), alkaline phosphatase (AlkPase), catalase (Cat), succinic dehydrogenase (SDH), glucose-6-phosphatase (G6Pase) and K+ p-nitrophenyl phosphatase (K+ pNPPase) were studied in the liver and kidney as histopathological biomarkers, to assess the effects of the two compounds in organs generally involved in the metabolism and excretion of different drugs. As oxidative stress may also develop as a consequence of the toxic effect of chemicals, reactive oxygen species (ROS) production was evaluated by a histochemical method. The results indicated that some enzyme activities and ROS expression changed in a dose-related manner. Nevertheless, neither in the liver nor in the kidney were dramatic toxic effects evident. By contrast, the variations of some enzyme activities (AlkPase, AcPase, Cat, K+ pNPPase) were interpreted as possible defensive mechanisms for tolerating high dosage of the compounds.

Revelation of a novel CLN5 mutation in early juvenile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) are relatively common storage diseases of childhood and early adolescence. Ultrastructural shape of storage cytosomes, type of disease gene, and age of onset serve to classify the different NCLs, some of which appear to cluster in Scandinavian countries. The CLN5 form usually presents as a classical epileptiform encephalopathy of late infancy but a more aggressive cognitive impairment has been described in a single family. We report two sibs harbouring a novel mutation (p.Tyr258Asp) in the CLN5 gene and displaying behaviour disturbances and mental deterioration, rather than epilepsy, as the dominant disease manifestation at onset.

Preparation and evaluation of nanoparticles made of chitosan or N-trimethyl chitosan and a cisplatin-alginate complex.

In this work, nanoparticles with a negative or positive surface charge were prepared through electrostatic interaction of an anionic cisplatin-alginate complex with a cationic polyelectrolyte, namely chitosan or N-trimethyl chitosan (substitution degree of 85%). Statistical experimental design allowed the study of the influence of component amounts on the characteristics of nanoparticles. Mean particle diameter ranged from 180 nm to 350 nm. After 24 h, while the cisplatin-alginate complex released almost all the drug in saline-buffered solution at pH 7.4, approximately 40% w/w of total cisplatin was released from negative nanoparticles and roughly 50% w/w from positive ones. The same cumulative amounts of released drug were found after 48 h, with a progressive reduction to lower values up to 6 days. Drug loading of nanoparticles with a positive zeta potential (43 mV-60 mV) ranged from 13% w/w to 21% w/w and particle yield, referred to total polymers, was about 15% w/w (50% w/w if referred to cisplatin-alginate complex). Nanoparticles with a negative zeta potential (-34 mV) were obtained with a yield of 40% w/w and a drug loading of 18% w/w. These nanoparticles were the least active on all cell lines tested, while the cytotoxic activity of the positive nanoparticles was similar to or lower than that of cisplatin, probably depending on the combination of sizes and zeta potential values, on P388 murine and A2780 human cells. On A549 human cells, the nanoparticles with the smallest size and the lowest positive zeta potential were more active than cisplatin and showed a similar capability in inducing apoptosis in A2780 human cells. These results indicate that cisplatin complexes with polycarboxylate polymers can be transformed into cisplatin particulate carriers of high potential interest.

Somatostatin analogues, a series of tissue transglutaminase inducers, as a new tool for therapy of mesenchimal tumors of the gastrointestinal tract.

Imatinib, a tyrosine kinase inhibitor directed against the enzymatic domain of KIT protein, was found to produce dramatic clinical responses in metastatic gastrointestinal stromal tumors (GISTs). However, resistance usually develops thus determining treatment failure. The present study was performed to analyse the expression of somatostatin receptor (SSTR) subtypes, modulators of tissue transglutaminase, in a series of GISTs and leiomyosarcomas by immunohistochemistry to identify a new potential therapeutic target. Sixteen cases (8 males and 8 females, age range: 38-73; 11 GISTs, 4 leiomyosarcomas, 1 leiomyoma) were studied. Immunohistochemical detection of the relevant SSTRs was performed on paraffin-embedded tissue sections, stained with polyclonal antibodies directed against the five somatostatin receptor subtypes. We found 7 out of 16 (44%) tumors expressing all SSTRs and 14 out of 16 (87%) tumors positive for at least 3 subtypes. SSTR2A was the most represented subtype in the tumors studied, being expressed in approximately 70% of cases exhibiting an intense labeling in most of these cases. The significant expression of SSTRs shown in this series of GISTs and gastrointestinal leiomyosarcomas suggests a potential therapeutic target to be explored alone and/or in combination with other therapeutic agents in the setting of refractory GI stromal tumors.

Immunomodulatory properties of cefaclor: in vivo effect on cytokine release and lymphoproliferative response in rats.

The proper and coordinated response of the host immune system to bacterial infections is known to play a central role in the eradication of an infection. Therefore, the impact of antibiotics on both innate and acquired host immunity may be involved in the therapeutic outcome. The aim of this study was to evaluate the effects of the widely used cephalosporin cefaclor on some parameters of the immune system in ex vivo conditions. The results demonstrated that short-term (3 to 6 days) treatment with this antibiotic induced pleiotropic modification of rat spleen cells upon ex vivo stimulation with the polyclonal mitogen PHA, entailing increased lymphoproliferative responses, augmented IFN-gamma, IL-2 and IL-10 synthesis and decreased production of IL-4 and IL-6 in comparison to spleen cells from control rats. The mononuclear spleen cells of healthy rats released larger amounts of IFN-gamma and IL-2 in culture supernatants in response to polyclonal mitogenic stimulation with PHA compared to the spleens of the control rats receiving vehicle only. Simultaneously, the treatment with cefaclor augmented PHA-induced lymphoproliferative responses and reduced the synthesis of IL-4 and IL-6. These data depict a type 1 cytokine inducing and immunostimulatory pharmacological profile that, by activating the innate and acquired immune system, would be synergistic with cefaclor antibacterial activity.

Optimizing ammonia processing conditions to enhance susceptibility of legumes to fiber hydrolysis: alfalfa.

An ammonia process was applied at several ammonia loadings, moisture contents, temperatures, and dwell times. A cellulase loading of 5 FPU/g dry matter and a 24 h incubation time were used to produce the sugars, which were measured as reducing sugars and by HPLC. Optimal processing conditions caused a 76% of theoretical yield (2.9-fold above untreated). Cellulose and hemicellulose conversions were 68 and 85% (vs 38 and 34% in untreated, respectively). The short hydrolysis time and relatively low enzyme loading suggests great potential to produce sugars from alfalfa.

Optimizing ammonia processing conditions to enhance susceptibility of legumes to fiber hydrolysis: Florigraze rhizoma peanut.

A warm-season legume, Florigraze rhizoma peanut (FRP), was used as the source of fiber to produce sugars. FRP was subjected to several ammonia-processing conditions using temperature, biomass moisture content, and ammonia loading as process variables during a 5-min treatment. A cellulase loading of 2 FPU/g DM and 24 h incubation were used to produce the sugars. Total sugar yield was 3.34-fold higher in the optimal treatment (1.5 g ammonia/g DM-60%-90 degrees C) compared to untreated and was 65.3% of theoretical. Cellulose and hemicellulose conversions increased from 30 and 15.5% in untreated FRP to 78 and 34% in treated FRP.

Optimizing ammonia pressurization/depressurization processing conditions to enhance enzymatic susceptibility of dwarf elephant grass.

An ammonia pressurization/depressurization process was investigated to evaluate the potential of producing reducing sugars from dwarf elephant grass, a warm-season forage. Moisture, temperature, and ammonia loading affected sugar yield (p < 0.0001). At optimal conditions, ammonia processing solubilized 50.9% of the hemicellulose and raised the sugar yield (percentage of theoretical) from 18 to 83%. Glucose and xylose production were increased 3.2- and 8.2-fold, respectively. The mild processing conditions of the ammonia treatment (90-100 degrees C, 5 min), the low enzyme loading (2 international filter paper units/g), and the short hydrolysis time (24 h), greatly enhance the potential of using forages to produce sugars valuable for several applications.

Correction of posterior crossbite in the primary dentition.

This study shows the types of malocclusions that can be treated in the primary dentition and advocates correction of maxillary constrictions from five years of age onwards, when such constrictions lead to crossbites. A preferred method to treat maxillary constrictions is through rapid maxillary expansion (RME) with the Haas expander, even in the early stages of occlusal development. This paper presents a cross-sectional evaluation and a long-term follow-up of the upper permanent incisors during the active and passive phases of RME and shows no signs of root or pulp alterations.

Massive lower gastrointestinal hemorrhage in patients with portal hypertensive enteropathy: a report of two cases.

Mucosal and vascular changes in the lower gastrointestinal tract occur commonly in patients with portal hypertension. Portal enteropathy, however, is usually asymptomatic, though occasionally clinically significant for chronic gastrointestinal bleeding. Massive hemorrhage has only rarely been described and its management is controversial. Even though more effective non-operative treatments are now available, an emergency porta-systemic shunt procedure remains an important option for selected patients. We report on two cases of massive lower gastrointestinal bleeding from portal hypertensive enteropathy secondary to post-viral cirrhosis.