Transendothelial migratory pathways of V delta 1+TCR gamma delta+ and V delta 2+TCR gamma delta+ T lymphocytes from healthy donors and multiple sclerosis patients: involvement of phosphatidylinositol 3 kinase and calcium calmodulin-dependent kinase II

We have previously reported that the Vdelta2(+)TCRgammadelta(+) T lymphocyte subset, expressing the NK receptor protein 1a (NKRP1a; CD161), is expanded in patients with relapsing-remitting multiple sclerosis and uses this molecule to migrate through endothelium. In this work, we show that Vdelta1(+) and Vdelta2(+) gammadelta T lymphocytes use distinct signal transduction pathways to accomplish this function. Indeed, we have found that Vdelta1(+) cells lack NKRP1a and selectively express the platelet endothelial cell adhesion molecule 1 (PECAM1; CD31), which drives transendothelial migration of this cell subset, at variance with Vdelta2(+) T cells, which are PECAM1 negative and use NKRP1a for transmigration. Interestingly, when Vdelta2(+) T cells were pretreated with two specific inhibitors of the calcium calmodulin-dependent kinase II KN62 and KN93, but not with the inactive compound KN92, the number of migrating cells and the rate of transmigration were significantly decreased. In turn, the phosphatidylinositol 3 kinase blockers wortmannin and LY294002 exerted a dose-dependent inhibition of Vdelta1(+) cell migration. Finally, NKRP1a and PECAM1 engagement led to activation of different signal transduction pathways: indeed, oligomerization of NKRP1a on Vdelta2(+) T cells activates calcium calmodulin-dependent kinase II, while occupancy of PECAM1 on Vdelta1(+) cells triggers the phosphatidylinositol 3 kinase-dependent Akt/protein kinase Balpha activation. These findings suggest that subsets of gammadelta T lymphocytes may migrate to the site of lesion in multiple sclerosis using two different signaling pathways to extravasate.     

Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

Analysis of tufted capuchin (Cebus apella) courtship and sexual behavior repertoire: changes throughout the female cycle and female interindividual differences

Sexual solicitations and initiative (proceptivity, sensu Beach [1976] Horm Behav 7:105-138) are important components of the sexuality of females of many primate species. In the tufted capuchin (Cebus apella), female proceptivity characterizes the species' mating system. In study 1, we defined and discussed 20 behavioral patterns based on the observation of 6 females and 5 males living in two social groups. In study 2, each behavior, including mounting activity, was quantitatively assessed during the periovulatory and nonperiovulatory cycle phases of 4 females, detected on the basis of urinary progestin levels (N = 20 ovulatory cycles, 5 for each female); moreover, we monitored changes in females' social interactions (agonism, grooming activity, and play). Nine of the behaviors typically used by the female during courtship and in sexual interactions showed a dramatic increase during the periovulatory phase. Though males mounted females at an apparently higher rate during the periovulatory than the nonperiovulatory phase, the difference was not significant. However, when adult male mounting is separated into those which occur within play and nonplay contexts, there is a significant periovulatory phase effect for mounts not associated with play. Females groomed adult males at the same rate throughout the cycle. Agonism and play did not show any phase effect; however, females' avoidance of adult males significantly increased during the periovulatory phase. Finally, each female made a statistically different use of the behavioral repertoire by performing some behaviors more than others. This variability among females during courtship calls for further research into whether it affects mating success.     

Differential microbial clearance and immunoresponse of Balb/c (Nramp1 susceptible) and DBA2 (Nramp1 resistant) mice intracerebrally infected with Mycobacterium bovis BCG (BCG)

In mice, the gene encoding Nramp1 (natural resistance-associated protein 1) exists in two allelic forms, differing for a point mutation. According to Nramp1 genotype, extensive literature documents a clear-cut distinction of inbred strains in two non-overlapping groups that phenotypically express resistance (Nramp1r) and susceptibility (Nramp1s) to systemic infections. Here, we provide evidence that Nramp1r (DBA/2) and Nramp1s (Balb/c) mice differently handle intracerebral infection with Mycobacterium bovis BCG. Distinct trends of microbial clearance from the brain and also different patterns of local immune responses occur, thus arguing on the involvement of Nramp1 gene product on the accomplishment of cerebral anti-mycobacterial defenses.     

Monitoring the effects of drug treatment in rat models of disease by serum protein analysis

In this review we list from literature investigations on rat serum proteins using electrophoretic techniques in connection with drug testing. From our own research work, we provide annotated two-dimensional maps of rat serum proteins under control and experimental conditions. Emphasis is on species-specific components and on the effects of acute and chronic inflammation. We discuss our project of structural proteomics on rat serum as a minimally invasive approach to pharmacological investigation, and we outline a typical experimental plan for drug testing according to the above guidelines. We then report in detail on the results of our trials of anti-inflammatory drugs on adjuvant arthritis, an animal model of disease resembling in many aspects human rheumatoid arthritis. We demonstrate a correlation between biochemical parameters and therapeutic findings and outline the advantages of the chosen methodological approach, which proved also sensitive in revealing "side effects" of the test drugs. In an appendix we describe our experimental protocol when performing two-dimensional electrophoresis of rat serum.     

Caspase-1 levels in biological fluids from patients with multiple sclerosis and from patients with other neurological and non-neurological diseases

In multiple sclerosis (MS), pathological white matter damage in the central nervous system is sustained by immune-inflammatory response. Caspase-1 plays a pivotal role in immune-mediated inflammation, as it regulates the cellular export of IL-1beta and IL-18. We carried out a preliminary in vitro study of the kinetics of extracellular caspase-1 release. We then measured caspase-1 levels in paired serum and cerebrospinal fluid (CSF) samples of 75 MS patients, 15 healthy subjects, and patients with other neurological diseases. Paired synovial fluid and serum samples of patients with juvenile idiopathic arthritis, and paired sputum and serum samples of asthma patients were also studied. Mean serum caspase-1 concentrations did not differ between groups. Caspase-1 was detected in the CSF of patients with acute, but not stable, MS [7.5 +/- (SEM) 0.9 pg/ml; test's sensitivity, 56% and specificity, 100%]. Its levels correlated with pleocytosis. The highest mean caspase-1 levels were found in the arthritic synovial fluids (945.5 +/- 126.6 pg/ml, which correlated with erythrocyte sedimentation rate), and in the sputum samples (370.1 +/- 71.0 pg/ml, which correlated with the number of macrophages in the sputum). On condition that caspase-1 is determined in the fluids pertaining to the disease-specific inflammatory sites, its level is a reliable marker of ongoing immune-inflammatory response. The enzyme measurement in CSF can also help define state-trait in MS.     

Molecular characterization of p53 mutations in primary and secondary liver tumors: diagnostic and therapeutic perspectives

p53 is one of the most mutated genes in human cancer. We have performed the molecular characterization of p53 and have searched for correlations with etiological factors and clinical parameters in primary and secondary liver tumors. A systematic study was carried out, innovative in many respects, to determine the mutational pattern of all 11 exons of p53 and analysis was extended also to exons 1–4 and 9–11 and the exon/intron junctions. Our analyses were performed on case histories of 114 patients from the European area and highlighted p53 mutation patterns different from those reported in the literature for the same tumors. In our case history, different tumors of the same organ showed a different frequency and distribution of mutations. In analyzed tumor types, gene status was a prognostic indicator of survival because patients undergoing liver resection without mutated p53 had a more favorable prognosis than mutated patients. This suggests p53 molecular diagnosis could become a further criterion in the decision for surgery and possible therapies. We describe the ideal conditions for polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequencing, which we have set in order to optimize yields, sensitivity, and time of what might become a massive molecular screening.     

An investigation of the seasonal pattern of mannitol content in deciduous and evergreen species of the oleaceae growing in northern Sicily

In several species of the Oleaceae, mannitol, already present at considerable levels, accumulates in response to stress. This family comprises both deciduous and evergreen species, and we investigated the role of mannitol in deciduous malacophyll and evergreen sclerophyll species growing under the same conditions in the field. The relationship between mannitol content and changes in rainfall or temperature was also studied. The mannitol content of leaves of Fraxinus ornus L., F. angustifolia Vahl., Olea europaea L. and Phillyrea media L. was determined by gas chromatography. Leaf samples were collected once a month for 1 year. In the two ash species, the seasonal pattern of mannitol content appeared the same: a gradual increase in spring, peaking in summer, followed by a gradual decrease. The mannitol content was similar in both species, ranging between 260 and 720 micromol g(-1) d. wt. The seasonal pattern of mannitol content in Olea and Phillyrea was similar for both species, but unlike that of Fraxinus did not show a summer peak. Rainfall was negatively correlated with the seasonal increase of mannitol content in ash. Mannitol content increased gradually during drought, reaching a maximum value at the end of the dry season. Temperature did not have a direct influence on mannitol content. In Olea and Phillyrea, variations in mannitol content were poorly correlated with rainfall or temperature, indicating that mannitol does not have a primary role in the response of these species to the hot, dry summer conditions.