Tacrolimus and Mycophenolate Mofetil Regimens in Transplantation

Immunosuppression, although necessary to enable the graft to escape the consequences of immune surveillance, carries some risks for the patient. There is an associated increase in neoplasms, opportunistic infections and end-organ toxicity. In addition, even with excellent patient compliance, rejection (acute and chronic) remains a major limitation that contributes to the loss or decrease in the function of the allograft. New drugs have been added to the armamentarium of immunosuppressive agents to suppress allograft rejection and to rescue grafts from cyclosporin-resistant rejection. With the availability of these immunosuppressive agents, it has become increasingly difficult to choose the appropriate combination of immunosuppressants with a beneficial effect for the patient and for the allograft. We describe 2 new immunosuppressive agents and some of their different uses in solid organ transplantation.     

HLA Antigens in 16 Families with Xeroderma Pigmentosum

Xeroderma pigmentosum is an autosomal recessive disease. HLA-A and -B typing was performed on peripheral blood lymphocytes and platelets. Sixteen Tunisian families were typed with 37 patients and 108 relatives. Genetic transmission of the disease and of the HLA system seemed to be independent in this study. Comparison of HLA gene frequencies between (unrelated) parents of patients and a control population showed no difference, proving that there is no clear association in populations between deleterious XP genes and a particular HLA gene. However, an excess of identical HLA among pairs of diseased siblings would suggest that the disease is polymorphic and a form of the XP could be linked to HLA.     

Nitrosative stress in the bronchial mucosa of severe chronic obstructive pulmonary disease

BACKGROUND: Reactive nitrogen species, formed via the reaction of nitric oxide (NO) with superoxide anion and via (myelo)peroxidase-dependent oxidation of NO(2)(-), have potent proinflammatory and oxidizing actions. Reactive nitrogen species formation and nitrosative stress are potentially involved in chronic obstructive pulmonary disease (COPD) pathogenesis. OBJECTIVES: To investigate the expression of markers of nitrosative stress, including nitrotyrosine (NT), inducible NO synthase (iNOS), endothelial NO synthase (eNOS), myeloperoxidase (MPO), and xanthine oxidase (XO) in bronchial biopsies and bronchoalveolar lavage from patients with mild to severe stable COPD compared with control groups (smokers with normal lung function and nonsmokers). METHODS: The expression of NT, iNOS, eNOS, MPO and XO in the bronchial mucosa and bronchoalveolar lavage of patients was measured by using immunohistochemistry, Western blotting, and ELISA and correlated with the inflammatory cell profile. RESULTS: Patients with severe COPD in stable phase had higher numbers of NT(+) and MPO(+) cells in their bronchial submucosa compared with mild/moderate COPD, smokers with normal lung function, and nonsmokers (P < .01). iNOS(+) and eNOS(+) but not XO(+) cells were significantly increased in smokers with COPD or normal lung function compared with nonsmokers (P < .05 and P < .01, respectively). In patients with COPD, the number of MPO(+) cells was significantly correlated with the number of neutrophils (r = +0.61; P < .0025) in the bronchial submucosa. Furthermore, the number of NT(+) and MPO(+) cells was negatively correlated with postbronchodilator FEV(1). CONCLUSION: These data suggest that nitrosative stress, mainly mediated by MPO and neutrophilic inflammation, may contribute to the pathogenesis of severe COPD.     

Partial protective effect of CCR5-Delta 32 heterozygosity in a cohort of heterosexual Italian HIV-1 exposed uninfected individuals

Despite multiple sexual exposure to HIV-1 virus, some individuals remain HIV-1 seronegative (exposed seronegative, ESN). The mechanisms underlying this resistance remain still unclear, although a multifactorial pathogenesis can be hypothesised. Although several genetic factors have been related to HIV-1 resistance, the homozigosity for a mutation in CCR5 gene (the 32 bp deletion, i.e. CCR5-Delta32 allele) is presently considered the most relevant one. In the present study we analysed the genotype at CCR5 locus of 30 Italian ESN individuals (case group) who referred multiple unprotected heterosexual intercourse with HIV-1 seropositive partner(s), for at least two years. One hundred and twenty HIV-1 infected patients and 120 individuals representative of the general population were included as control groups. Twenty percent of ESN individuals had heterozygous CCR5-Delta 32 genotype, compared to 7.5% of HIV-1 seropositive and 10% of individuals from the general population, respectively. None of the analysed individuals had CCR5-Delta 32 homozygous genotype. Sequence analysis of the entire open reading frame of CCR5 was performed in all ESN subjects and no polymorphisms or mutations were identified. Moreover, we determined the distribution of C77G variant in CD45 gene, which has been previously related to HIV-1 infection susceptibility. The frequency of the C77G variant showed no significant difference between ESN subjects and the two control groups.     

Fluorescence in situ hybridization analysis of HER-2/neu in brushings of normal oral mucosa

Oncogene alterations have been clearly demonstrated to be related to the carcinogenesis and progression of oral squamous cell carcinoma (OSCC). However, the analysis of these alterations for screening and early diagnostic purposes generally requires invasive techniques for surgical removal of pathological epithelium. The aim of the present study was to assess the feasibility of fluorescence in situ hybridization (FISH) analysis of HER-2/neu amplification in oral mucosa brushings and to compare the HER-2/neu status with the history and smoking and drinking habits of healthy subjects. Cells obtained by centrifugation of oral brushings from 21 subjects (overall no. of cells: 5125) were suspended in physiological saline and fixed onto two slides for cytological evaluation and FISH analysis (dual-target, dual-color fluorescence assay) of the HER-2/neu gene and CEP17 centromere. A mean of 89.8% of the cells showed two HER-2/neu signals and a mean of 94% had two CEP17 signals at fluorescent microscopy. Finally, a mean of 96% of cells with HER-2/neu / CEP17 had a ratio equal to 1. No association between smoking and drinking habits, age and the HER-2/neu and CEP17 characteristics evaluated by FISH was found.     

L-arginine, the precursor of nitric oxide, abolishes the effect of estrogens on bleeding time in experimental uremia.

We have reported previously that conjugated estrogens that are effective in shortening the prolonged bleeding time in uremic patients are also effective on bleeding time in a rat model of uremia. Using such a rat model we have recently demonstrated that nitric oxide (NO), an endothelium-derived vasodilator, is involved in mediating the bleeding tendency of uremia. With the present study we wanted to investigate whether conjugated estrogen mixture or its active component, 17 beta-estradiol, reduce uremic bleeding by interfering with the NO pathway. Our results showed that the shortening effect of conjugated estrogen and 17 beta-estradiol on bleeding time of uremic rats was completely reversed by giving the animals the NO precursor L-arginine, but not D-arginine, which is not a precursor of NO. Dexamethasone which at variance to progesterone inhibits the process of induction of NO-forming enzyme, shortened the prolonged bleeding time of uremic rats within 4 hours from injection. This effect was eliminated by L-arginine but not D-arginine administration. The glucocorticoid receptor antagonist cortexolone prevented the shortening of bleeding time induced by dexamethasone, suggesting that a receptor-mediated mechanism is involved in the hemostatic effect of dexamethasone as previously reported for estrogens. Unlike conjugated estrogens and dexamethasone, progesterone had no effect on bleeding time. All these findings would indicate that the effect of estrogens and dexamethasone on primary hemostasis in uremia might be mediated by changes in NO synthetic pathway.     

Endoplasmic reticulum stress and unfolded protein response in inclusion body myositis muscle

Proteins in the endoplasmic reticulum (ER) require an efficient system of molecular chaperones whose role is to assure their proper folding and to prevent accumulation of unfolded proteins. The response of cells to accumulation of unfolded proteins in the ER is termed "unfolded protein response" (UPR). UPR is a functional mechanism by which cells attempt to protect themselves against ER stress, resulting from the accumulation of the unfolded/misfolded proteins. Because intracellular inclusions, containing either amyloid-beta (Abeta) or phosphorylated tau, are the characteristic feature of sporadic inclusion body myositis (s-IBM) muscle biopsies, we studied expression and immunolocalization of five ER chaperones, calnexin, calreticulin, GRP94, BiP/GRP78, and ERp72, in s-IBM and control muscle biopsies. Physical interaction of the ER chaperones with amyloid-beta precursor protein (AbetaPP) was studied by a combined immunoprecipitation/immunoblotting technique in s-IBM and control muscle biopsies, and in AbetaPP-overexpressing cultured human muscle fibers. In all s-IBM muscle biopsies, all five of the ER chaperones were immunodetected in the form of inclusions that co-localized with amyloid-beta. By immunoblotting, expression of ER chaperones was greatly increased as compared to the controls. By immunoprecipitation/immunoblotting experiments, ER chaperones co-immunoprecipitated with AbetaPP. Our studies provide evidence of the UPR in s-IBM muscle and demonstrate for the first time that the ER chaperones calnexin, calreticulin, GRP94, BiP/GRP78, and ERp72 physically associate with AbetaPP in s-IBM muscle, suggesting their playing a role in AbetaPP folding and processing.     

Aspirin and the prevention of experimental arterial thrombosis: difficulty in establishing unequivocal effectiveness

A trial of the efficacy of aspirin in the prevention of thrombotic occlusion of an "aortic loop" in rats was made simultaneously by two experimental surgeons. A relatively large dose of aspirin (80-100 mg/kg/day) was used, starting two days before operation. It appeared that aspirin was of limited benefit, reducing thrombotic occlusions by about 17% seven days after the insertion of the loop into the abdominal aorta. Although the average occlusion time was prolonged by about 17% in aspirin-treated animals, the separate trials gave no conclusive result. When the data from both operators were pooled, a statistically significant protection by aspirin was apparent (p = 0.02), by a two-tailed Student's t test. However, on using the powerful non-parametric randomization test, the occlusion times in control and aspirin-treated groups appeared not statistically different (p = 0.07). No significant difference was also found between control and treated groups when data were analyzed by X2 test. Independently of the statistical analysis, these data are quite similar to those obtained from aspirin trials in men surviving myocardial infarction. This finding points to the usefulness of the aorta loop as an animal model for arterial thrombosis.     

Aspirin kinetics and inhibition of platelet thromboxane generation-relevance for a solution of the "aspirin dilemma"

Six healthy male volunteers received aspirin (ASA) in a compressed (320 mg) and an enteric-coated (800 mg) formulation as single oral doses ten days apart. Ten plasma samples were obtained from each volunteer between 5 and 120 min after compressed ASA, and seven between 10 and 240 min after enteric-coated ASA. ASA was undetectable (less than 100 ng/ml) in plasma from three subjects receiving compressed ASA and two receiving the enteric-coated preparation. Plasma levels and kinetic parameters of salicylate were the same in subjects with undetectable and detectable ASA plasma levels. More than 98% inhibition of pre-drug serum TXB2 was noted in all samples collected one and four hours after either ASA preparation. TXB2 generation recovered on average by 3.5% at 24 hr with both preparations. Thus inhibition of platelet TXB2 generation occurred independently of the amount of ASA reaching the peripheral circulation. If this is due to inhibition of platelet function in the enterohepatic circulation followed by extensive first-pass deacetylation of ASA, vascular PGI2 synthesis could be spared. A better knowledge of the kinetic parameters of ASA for each of the formulations used in thrombosis prevention trials might help in solving the "aspirin dilemma".