Cyclophosphamide modulates CD4+ T cells into a T helper type 2 phenotype and reverses increased IFN-gamma production of CD8+ T cells in secondary progressive multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system considered to be mediated by T helper type-1 cells. Several agents have been found to modify the disease course of MS, including interferon-beta1 (IFN-beta1), glatiramer acetate mitoxantrone. We have employed pulse therapy with cyclophosphamide in a selected group of patients with actively progressive disease. Chemokine receptors have been found to differentiate between polarized T helper type-1 (Th1) and type-2 (Th2) lymphocytes. The chemokine receptors CCR5 and CXCR3 are expressed primarily on Th1 cells and CCR3, CCR4 and CCR8 on Th2 cells. Previous studies of the expression of chemokine receptors in MS have shown that active MS plaques are infiltrated by CCR5(+) and CXCR3(+) T cells. Some of these T cells may express both CCR5 and CXCR3. These T cells are major producers of IFN-gamma, which worsens the clinical condition of patients with MS. We previously found that patients with MS had a high proportion of CXCR3(+) T cells and that those with chronic progressive MS had a high proportion of CCR5(+) T cells in their peripheral blood. We report here that in patients with secondary progressive MS, cyclophosphamide induces a marked increase in the percentage of CCR4(+) T cells that produce high levels of IL-4 and reverses the increase in the percentages of IFN-gamma-producing CCR5(+) and CXCR3(+) CD8(+) T cells. Furthermore, therapy with cyclophosphamide increases IL-4-producing CD4(+) T cells and reverses the increase in IFN-gamma-producing CD8(+) T cells. Our study shows that cyclophosphamide has immunomodulatory properties besides its suppressive effects, and that chemokine receptors can be important tools both for understanding the immune dysregulation in MS and for monitoring response to therapy.     

Comparison of kinetics of induction of DNA adducts and gene mutations by a nitrofuran compound, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), in the caecum and small intestine of Big Blue mice

In previous experiments, i.p. injection of the 5 nitronaphthofuran derivative 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000) to lacI transgenic Big Blue mice led to an increase in the mutant frequency (MF), especially in the caecum and the small intestine. In the present work, the in vivo genotoxicity of R7000 in these two target organs was further investigated. Big Blue mice were treated with a single daily i.p. injection of R7000 of 0.05-0.5 mg/day for five consecutive days and killed 28 days later. These treatments led to significant increases in MF of 1.8-, 3- and 5.4-fold at 0.1, 0.2 and 0.5 mg/day R7000, respectively, in the small intestine. In the caecum, a mutagenic effect, of 4.5-fold, was only observed at the highest dose. DNA adduct formation and MFs resulting from R7000 were also analysed in parallel at various times after the last injection. R7000 led to 14 and seven different nucleotide modifications in the caecum and small intestine, respectively. Three hours after the final injection the level of induced DNA adducts was 10 times higher in the caecum than in the small intestine. From 3 h to 5 days after the final injection, 93 and 58% of DNA adducts disappeared in the caecum and small intestine, respectively. The resulting MF values were similar when comparing the two organs. Analysis of the R7000-induced mutation spectrum in the caecum showed that single G:C and large, > or =3 bp deletions and GC-->CG transversions were the first induced mutations at the end of the treatment. Fifteen days later, the R7000 mutation specificity characteristics already reported in Escherichia coli and in the small intestine of Big Blue mice were evident in the caecum, with the two major events being GC-->TA transversions and deletions of one G:C base pair. In both organs, a relationship between the decrease in R7000-DNA adducts and induction of MF was evident. However, the efficiency of this compound in damaging DNA was not correlated with the capacity of DNA lesions to lead to mutations. Some discrepancies in the R7000 genotoxic effects between the two organs were observed, which may be attributable to differences in the metabolic activation pathway of the compound, as well as to DNA repair proficiency in each tissue.     

Facial appearance in persistent hyperinsulinemic hypoglycemia

Persistent hyperinsulinism is the most common cause of recurrent hypoglycemia in infancy because of inappropriate oversecretion of insulin by the pancreas. Pancreatic lesions can be either focal or diffuse, and they have distinct molecular bases. We have studied the facial features in 17 unrelated patients presenting with neonatal (n = 8) or infancy-onset (n = 9) hyperinsulinism. Hyperinsulinism was related to focal adenomatous hyperplasia (n = 7), diffuse hyperinsulinism (n = 5), non-operated hyperinsulinism (n = 2), and hyperinsulinism with hyperammonemia (n = 3). SUR1 or Kir6.2 mutations were found in six of seven focal adenomatous hyperplasia and three of five diffuse hyperinsulinism. A loss of the maternal allele from chromosome 11p15 in the lesion was found in all focal adenomatous hyperplasia. GLUD1 mutations were found in all patients with hyperammonemia. Large birth weight (mean > 3,800 g) was consistently observed (11/17) but protruding tongue, exomphalos, or visceromegaly were never noted and Wiedemann-Beckwith syndrome could always be ruled out. All patients presented with high forehead, small nasal tip, and short columella giving the impression that the nose is large and bulbous, smooth philtrum, and thin upper lip. A square appearance to the face was more obvious in younger patients. These specific facial features, observed in patients with hyperinsulinism of various molecular mechanisms, could be the consequence of fetal intoxication by insulin. However, to date, facial anomalies have not been noted in infants of diabetic mothers and inversely, malformations that are commonly reported in infants of diabetic mothers were not present in our hyperinsulinemic patients.     

Increased incidence of mitochondrial cytochrome c-oxidase gene mutations in patients with myelodysplastic syndromes

Mitochondria (mt) play an important role in both apoptosis and haem synthesis. The present study was conducted to determine DNA mutations in mitochondrial encoded cytochrome c-oxidase I and II genes. Bone marrow (BM) biopsy and aspirate, peripheral blood (PB) and buccal smear samples were collected from 20 myelodysplastic syndrome (MDS) patients and 10 age-matched controls. Cytochrome c-oxidase I (CO I) and II (CO II) genes were amplified using polymerase chain reaction and sequenced. CO I mutations were found in 13/20 MDS patients and the CO II gene in 2/10 normal and 12/20 MDS samples, irrespective of MDS subtype. Mutations were substitutional, deletional and insertional. CO I mutations were most common at nucleotide positions 7264 (25%) and 7289 (15%), and CO II mutations were most common at nucleotide positions 7595 (40%) and 7594 (30%), suggesting the presence of potential 'hot-spots'. Mutations were not found in buccal smears of MDS patients and were significantly higher in MDS samples compared with age-matched controls in all cell fractions (P < 0.05), with bone marrow high-density fraction (BMHDF) showing a higher mutation rate than other fractions (P < 0.05). MDS marrows showed higher levels of apoptosis than normal controls (P < 0.05), and apoptosis in BMHDF was directly related to cytochrome c-oxidase I gene mutations (P < 0.05). Electron microscopy revealed apoptosis affecting all haematopoietic lineages with highly abnormal, iron-laden mitochondria. These results suggest a role for mt-DNA mutations in the excessive apoptosis and resulting cytopenias of MDS patients.     

Effect of simultaneous and/or consecutive administration of the broad spectrum anthelmintic flubendazole together with praziquantel in experimental Schistosoma mansoni infection

This study is a trial to demonstrate the effect of the broad spectrum anthelmintic drug flubendazole (methyl 5-(p-fluoro-benzoyl)-2-benzimidazolecarbamate, CAS 31430-15-6), a mebendazole derivative, together with praziquantel (CAS 55268-74-1, EMBAY 8440, Biltricide) in murine schistosomiasis mansoni. Moreover, the relationship between the posttreatment worm burden, oogram pattern, tissue egg load and hepatic granuloma volume was also investigated. Three main groups of Swiss albino mice infected with Schistosoma mansoni cercariae were used in the experiment. Group I included infected untreated control mice. Group II: Subgroup II (a): Animals received 1/3 the dose of praziquantel 25 days post infection. Subgroup II (b): Mice were given 1/3 dose of flubendazole 25 days post infection. Subgroup II (c): Animals received the combination (1/3 dose of flubendazole + 1/3 the dose of praziquantel 25 days post infection. Group III: Subgroup III (a): Mice were given 1/3 the dose of praziquantel 7 weeks post infection. Subgroup III (b): Mice received 1/3 dose of flubendazole 25 days post infection. 24 days later, 1/3 the dose of praziquantel was given. Mice given the consecutive drug regimen (flubendazole 1/3 single oral dose 25 days post infection, then praziquantel 1/3 oral dose for two successive days 24 days later, revealed a significant reduction in the recovery of adult schistosomes after portal perfusion (95.9%), absence of immature stages of ova development, a higher level of dead ova in the oogram and the smallest granuloma mean diameter. These data were less conspicuous in mice given the simultaneous drug regimen.     

Iron-deficiency anemia and protein-energy status in children aged 6 to 59 months in Tunisia

Our study is going to analyse the foodstuffs consumption of the people who have an iron deficiency and then compare it to the consumption of the people who don't have a deficiency. The anemic children who suffer from an iron deficiency have shown an average supply of iron inferior to the required needs which is of 86%. 22.7% of these children have an available iron supply below the recommended average which represents the limit of a severe deficiency risk. No child exceed the severe deficiency risk limit in proteins. 31% of the deficient children have daily energetic supplies inferior to the minimum limit of the daily-required supplies and they are on the brink of a deficiency. The protein needs are common in children but not the energetic ones.     

Fluorescence resonance energy transfer reveals a binding site of a photosensitizer for photodynamic therapy

Phthalocyanine (Pc) 4, like many photosensitizers for photodynamic therapy (PDT), localizes to intracellular membranes, especially mitochondria. Pc 4-PDT photodamages Bcl-2 and Bcl-xL, antiapoptotic proteins interacting with the permeability transition pore complex that forms at contact sites between the inner and outer mitochondrial membranes. These complexes and the inner membrane are unique in containing the phospholipid cardiolipin. Nonyl-acridine orange (NAO) is a specific probe of cardiolipin. Here we show evidence for fluorescence resonance energy transfer from NAO to Pc 4, defining a binding site for the photosensitizer. This observation establishes an innovative tool for exploring the localization of other photosensitizers and additional fluorescent, mitochondrion-localizing drugs having appropriate spectral properties.