Immunomodulatory properties of porcine, bone marrow-derived multipotent mesenchymal stromal cells and comparison with their human counterpart.

Thanks to their immunonodulatory properties, multipotent mesenchymal stromal cells (MSCs) are a promising strategy for preventing/reducing the risk of graft rejection after hematopoietic cell and solid organ transplantation. We have previously demonstrated that porcine MSCs (pMSCs) can be isolated from bone marrow and display similar morphology and differentiative capacity as compared to human MSC (hMSCs). In this study, we investigated the in vitro immunomodulatory properties (namely the ability to suppress lymphocyte proliferation in response to phytohemagglutinin and the cytokine production in the culture supernatants) of pMSCs from six Large White 6-month old piglets. Similarly to hMSCs, pMSCs reduced the phytohemagglutinin-induced lymphocyte proliferation. High levels of IL-6 were found in culture supernatants, whereas IL-10 and TGF-ß were not detectable. In conclusion, ex vivo expanded pMSCs share selected biological/functional properties with hMSCs. pMSCs may be used in in vivo models to investigate novel approaches of prevention of graft rejection in solid organ transplantation.

Phenotypic and genotypic alterations characterize patients bearing plasma cell dyscrasias with a high M-component.

As at present only a long-term follow-up can fully determine whether monoclonal gammapathies of undetermined significance (MGUS) will evolve into multiple myeloma (MM), this study attempted to identify other variables connected with the amount of monoclonal component (MC), generally considered as the most reliable marker of malignant evolution. Thirty-four MGUS subjects showing a high MC (> or = 15.0 g/l) but without clinical evidence of MM (MGUS group b), were characterized for their phenotypic and genotypic profile by comparing them either with 40 MM patients or with 24 subjects affected by a benign form of monoclonal gammapathy (MGUS group a) according to the standard criteria. In addition to the usual laboratory markers, the levels of expression of a panel of CD membrane subsets were measured on B and T lymphocytes. Also, the serum level of the p53 mutant protein and the structural alterations of the c-myc oncogene were evaluated. The results show that for MGUS group b patients, an increased M-protein was accompanied by significantly increased levels of peripheral blood CD3+ T cells and oncogenetic aberrations in c-myc. Since a high serum MC level seems to indicate a greater likelihood of malignant transformation for MGUS patients, these findings suggest that this relationship may be a result of the concomitant alterations observed at a phenotypic and genotypic level. Such alterations may be potentially useful as surrogate markers for the transition of benign to malignant (MM) plasma cell dyscrasia.

Serum interleukin-10 levels in patients affected with multiple-myeloma - correlation with the monoclonal component and disease progression.

Using a commercially available, competitive ELISA kit based on a polyclonal anti-interleukin-10 antibody, serum interleukin-10 (IL-10) levels were quantified in samples of different groups of patients: 20 healthy controls (CTR), 11 monoclonal gammopathies of uncertain significance (MGUS), 17 multiple myelomas (MM), 10 cancer patients (CANCER), 13 cancer patients + MGUS (MGUS-CA) and 7 MGUS patients after surgical removal of concomitant cancer (MGUS-SRCC). Results show significant differences of both the median levels of IL-10 and the monoclonal component (MC) in CTR, MGUS and MM (patients with increasing concentrations in the mentioned order). The IL-10 levels found in the three groups of cancer patients showed serum levels higher than those observed in the controls. Moreover, the surgical cancer removal was related to an IL-10 decrease. A higly significant correlation between serum IL-10 levels and the corresponding MC was also found in the MM-bearing patients and to a lesser extent, in MGUS patients, indicating that serum IL-10 is parallel to the amount of the activated clone causing the monoclonal gammopathy. Since human myeloma lines, cultured in vitro may release significant amounts of IL-10, the data presented support the hypothesis that serum IL-10, measured in myelomatous patients may, at least in part, derive from the activated clone causing the monoclonal gammopathy.

Autoimmune thrombocytopenic purpura in pregnancy: maternal risk factors predictive of neonatal thrombocytopenia.

Pregnancy in ATP women is not unusual. The problem of this association concerns the possibility of disease transmission to the fetus due to the crossing of maternal antiplatelet antibodies through the placenta. Maternal risk factors predictive of neonatal thrombocytopenia, can be identified as follows: severe thrombocytopenia, previous splenectomy, high titre of PA-IgG and/or SPB-IgG. In 63 pregnancies in ATP patients, we have evaluated whether the above maternal risk factors, considered in the third trimester, can provide useful criteria for the prediction of neonatal thrombocytopenia. In the third trimester, the distribution of maternal risk factors was as follows: 0 in 7 cases, 1 in 27 cases, 2 in 15 cases, 3 in 12 cases, 4 in 2 cases. From a statistical evaluation, the neonatal platelet values and the maternal risk factors seem inversely correlated (r -0.437; p = 0.0005). In particular, neonatal and maternal platelet count correlated positively (r = 0.249; p = 0.025); moreover, neonatal platelet count correlated negatively with Splenectomy (r = -0.209; p = 0.05), PA-IgG (r = -0.401; p < 0.0005) and SPB-IgG (r = -0.338; p < 0.005). We tried to apply a multiple regression model for all the above parameters which appears statistically significant (p = 0.001); the variability was about 30%. This regression model could be validated if applied to a larger number of cases, and it could represent an alternative to the invasive methods used for the diagnosis of neonatal thrombocytopenia.

Detection of Helicobacter pylori carriers by discriminant analysis of urea and pH levels in gastric juices.

An alternative approach to the problems inherent in current methods for detecting Helicobacter pylori carriers--that of being generally time-consuming, expensive, and not sufficiently sensitive--was devised by using the urea concentration and pH levels of gastric juices. A linear discriminant analysis of these variables, measured in 54 patients submitted to digestive endoscopy for gastritis, provided a mathematical formula for assigning the subjects (previously classified by other standard methods) to groups of either positive or negative H pylori carriers. The results obtained showed a correct classification in 52 out of 54 cases with only one false negative and one false positive case.

Influence of paradoxical sleep deprivation on the intermediate stage of sleep in the rat.

In the rat, paradoxical sleep (PS) is preceded by a short-lasting stage characterized by high amplitude cortical spindles and low frequency hippocampal theta rhythm. This intermediate stage (IS) is massively extended at the expense of PS by barbiturates and benzodiazepines. To further study the relationship between the IS and PS, six rats were PS deprived for 48 h by sitting on one of three small platforms surrounded by water. A control group of six other rats remained in dry cages with shavings. After 24 h PS deprivation the latency of IS occurrence increased, was unchanged after 48 h deprivation and decreased during recovery on shavings after 52 h deprivation. The total IS duration, number of episodes and mean duration per episode were unchanged during deprivation but increased during recovery. This recovery was characterized by an increase in the number of PS episodes and the total duration while the latency of PS occurrence decreased. The frequency of theta rhythm, unchanged for PS, increased for the IS during deprivation and recovery. In conclusion, the IS did not substitute for PS after selective deprivation.

Sleep cycle disturbances induced by apamin, a selective blocker of Ca(2+)-activated K+ channels.

Intracerebroventricular injections of low doses of apamin, a specific blocker of a class of Ca(2+)-activated K+ channels, induced insomnia and a long-lasting suppression of deep slow sleep and paradoxical sleep. Injected animals showed a late but important rebound of paradoxical sleep. Even after the recovery of a normal sleep amount, the circadian cycle remained disturbed during all the recording duration (96 h).

Influence of a GABA(B) receptor antagonist on the sleep-waking cycle in the rat.

The influence of CGP 35348 (a GABA(B) receptor antagonist) on the sleep-waking cycle was studied in rats. The animals were injected i.p. at the beginning of the light period and the data expressed by 2-h periods and total duration (6 h). At 100 mg/kg, slow-wave sleep (SWS) was decreased during the 6-h recording with a peculiar decrease during the first 2 h. SWS was subdivided into three stages: slow-waves; spindles occurring as SWS deepens; and intermediate stage appearing prior to paradoxical sleep (PS). Only the slow-wave stage and intermediate stage were decreased. Waking was increased during the 6-h recording. It was subdivided into waking with hippocampal theta rhythm (psychomotor active waking) and waking without theta activity (quiet waking). Both were increased during the first 2 h. However, quiet waking was increased throughout the recording duration. At 300 mg/kg, SWS was decreased during the three 2-h periods. This decrease was principally related to a decrease of the slow-wave stage. PS was increased over the 6-h recording with a marked increase during the second 2-h period. Consequently, under the influence of the GABA(B) receptor antagonist, the SWS was decreased at the expense of behavioral stages with cortical low-voltage activity (waking and PS). GABAergic neurons are present in the mesopontine structures responsible for these two stages. We can conclude that endogenous GABA acting at the GABA(B) receptor level participates in the regulation of waking and PS.

The brain response to the bee venom peptide MCD. Activation and desensitization of a hippocampal target.

The mast cell-degranulating peptide (MCD) isolated from bee venom has been found previously to have receptor sites in rat brain. Behavioral and electrocorticographic responses following intracerebroventricular injections of various doses of MCD have been analyzed. MCD produced a quasi-permanent hippocampal theta rhythm in the motionless animal alternating with epileptiform spike waves and paroxystic seizures. At a dose of 70 pmol seizures occurred for half of the treated rats. At a dose of 100 pmol generalized paroxystic crises were observed for all the rats. These effects were not antagonized by naloxone, morphine, diazepam and progabide. Rats recovered 24 h after a 100 pmol injection of MCD. A second ipsilateral injection to these rats showed the occurrence of a desensitization phenomenon. Desensitization was not observed when the second injection was contralateral. These physiological responses were studied in relation with a biochemical approach on membrane sites of action of MCD using [125I]MCD and their behavior in the desensitization process. The target of [125I]MCD is the ipsilateral hippocampus. Recovery from MCD effects was not due to MCD degradation. Desensitization was not due to down-regulation of the MCD receptor level.

Subtypes of K+ channels differentiated by the effect of K+ channel openers upon K+ channel blocker-induced seizures.

Intracerebroventricular injection of mast-cell degranulating peptide (MCD), dendrotoxin I (DTXI) and 4-aminopyridine (4-AP), 3 blockers of a subclass of K+ channel, produces seizures and convulsions. Three different K+ channel openers are potent blockers of MCD-induced hyperexicitatory effects when they are administered preventively but they are unable to inhibit the epileptogenic effects induced by DTXI and 4-AP which were thought to block the same K+ channel which is blocked by MCD.

Analogies and differences in the mode of action and properties of binding sites (localization and mutual interactions) of two K+ channel toxins, MCD peptide and dendrotoxin I.

Both the bee venom toxin, mast cell degranulating (MCD) peptide, and the snake toxin, dendrotoxin 1 (DTX1) induce epileptiform activity and paroxystic seizures after intracerebroventricular (i.c.v.) injection to rats. Although many of the properties of the two toxins, which are blockers of the same K+ channel, appear to be very similar, a number of differences have been found. (1) Induced seizures have an hippocampal origin for MCD and two different origins, situated in the cortex and in the limbic system, for DTX1. (2) A first i.c.v. administration of DTXI desensitizes against a second ipsilateral injection of the same peptide as we had previously observed for MCD. However no cross-desensitization was observed between the two different toxins. (3) The number of high affinity (Kd = 41 pM) binding sites for 125I-DTXI in synaptic membranes is about 5 times higher than the number of high affinity (Kd = 158 pM) binding sites for 125I-MCD. (4) Autoradiographic analysis of the distribution of high affinity 125I-DTX1 binding sites has been compared to our previous analysis of high affinity 125I-MCD binding sites. High levels of high affinity binding sites for both toxins seem to be localized in synapse-rich areas. However high affinity binding sites for the two toxins are not always co-localized. Analysis of the mutual interactions between DTXI and MCD binding sites has revealed the presence of classes of low affinity binding sites for MCD. In most areas of the brain, a large proportion of high affinity binding sites for DTXI is allosterically related to low affinity binding for MCD.

Ca2+ channel blockers prevent seizures induced by a class of K+ channel inhibitors.

Intracerebroventricular injection into rats of mast-cell degranulating peptide (MCD), dendrotoxin I (DTXI) and 4-aminopyridine (4-AP), three blockers of a subclass of K+ channels, elicited epileptiform wave bursts and convulsions. Three different types of L-type Ca2+ channel inhibitors (+)PN 200-110, a 1,4-dihydropyridine, (-)D888, a phenylalkylamine, and fluspirilene, a diphenylbutylpiperidine, were potent blockers of the convulsant-induced hyperexcitatory effects when they were administered preventively. D-AP5, a N-methyl-D-aspartate antagonist, was active on the 4-AP-induced seizures but was without effect on the MCD- and dendrotoxin-induced seizures.

Riluzole prevents hyperexcitability produced by the mast cell degranulating peptide and dendrotoxin I in the rat.

Using electroencephalographic (EEG) recordings in freely moving rats and extracellular neuronal firing-rate recordings in hippocampal slices, we examined the effects of riluzole (RP 54274), a compound with anti-glutamate properties, against the convulsive seizures and the cellular hyperexcitability produced by the mast-cell degranulating peptide (MCD), dendrotoxin I (DTXi) and 4-aminopyridine (4-AP). I.c.v. administration of riluzole (10 nmol) prevented the seizures induced by MCD, and to a lesser extent those due to DIXi, whilst leaving 4-AP seizures unaffected. This effect was also present after oral administration of the compound (4 mg kg-1) and lasted for approximately 6 h. Electrophysiological recordings in vitro confirmed that riluzole dose dependently and reversibly abolished the sustained increase in firing rate induced by both MCD and DTXi in the hippocampus. These results indicate that the anti-epileptic spectrum of riluzole in this model has similarities with, but is not identical to, that of classical potassium channel openers, and differs from that of calcium channel blockers or other glutamate antagonists such as D(-)-2-amino-5-phosphono-valeric acid. However, since MCD releases glutamate, the preventive effect of riluzole in this model may involve direct or indirect interaction with glutamatergic processes.

Incidence of chromosomes 1 and 17 aneusomy in breast cancer and adjacent tissue: an interphase cytogenetic study.

BACKGROUND: Characterization of the biopathologic events underlying the early steps of breast carcinogenesis may have a dramatic impact on reducing breast cancer mortality. Genes involved in breast tumorigenesis are localized on chromosomes 1 and 17, and numeric aberrations of these chromosomes have been correlated with breast cancer tumorigenesis and progression. According to the field cancerization hypothesis, specific chromosome aberrations may be present in breast cancer and in normal-appearing adjacent tissue. The latter changes reflect the genomic damage that follows longterm carcinogenic exposure and precede the morphologically detectable neoplastic transformation. We hypothesize that detection of these aberrations in benign breast epithelium may provide a tool for molecular risk assessment. STUDY DESIGN: Using fluorescence in situ hybridization with centromere-specific probes, we determined the status of chromosomes 1 and 17 in fresh imprints of 28 samples of primary tumors and 54 samples of their surrounding uninvolved parenchyma taken from patients undergoing operations for breast carcinoma. Ten contralateral breast biopsy specimens collected from patients with previous breast carcinoma were also evaluated as a surrogate of a high-risk group to rule out the hypothesis that chromosomal aneusomy in tumor-adjacent tissue could be related to a paracrine effect of the primary tumor. Ten samples of benign breast tissue taken from patients at low risk were used as controls to define tolerance limits for aneusomy definition. RESULTS: Using threshold values of 40% of signal loss and 13% of signal gain to define chromosome aneusomy (ie, mean + 3 SDs of the control group signals), we found the following: 1) almost all primary breast tumors were aneusomic for chromosomes 1 and 17; 2) primary breast tumor and adjacent uninvolved parenchyma shared the same pattern of chromosomes 1 and 17 aneusomy in 66.7% of patients; and 3) chromosomes 1 and 17 aneusomies in contralateral benign breast samples from high-risk patients were not different from those in primary breast tumor or adjacent tissue samples. CONCLUSIONS: These results suggest that chromosomes 1 and 17 aneusomy may represent an intermediate biomarker of breast tumorigenesis potentially useful to detect patients at high risk of breast carcinoma who may benefit from preventive interventions.

Fetal serotoninergic transplants in the fourth ventricle of adult rodents reverse sleep disturbances induced by neonatal administration of 5,7-dihydroxytryptamine.

Rats received a neonatal (at 4 days of age) intracisternal injection of 5,7-dihydroxytryptamine which eliminates serotonin (5-HT) throughout adulthood. Sleep recordings, performed on these rats at adult age, demonstrated significant decreases in paradoxical sleep. Dissociated fetal 5-HT cell suspensions transplanted in the IVth ventricle of these rats restored paradoxical sleep. However, fetal neuronal noradrenergic or cholinergic transplants did not restore paradoxical sleep. These results suggest that paradoxical sleep is directly or indirectly mediated through serotoninergic mechanisms.

Sleep-waking cycle in chronic rat preparations with brain stem transected at the caudopontine level.

The brain stem of rats was transected at the middle of the nucleus reticularis pontis caudalis. The preparations were maintained 2-9 days, and their EEG activity and behavior were studied. Maintained EEG activity and EEG arousal to visual and olfactory stimuli indicated the presence of sleep-waking cycle. Three stages were identified. Two of them corresponded to waking with hippocampal theta rhythm and to slow wave sleep in intact rats. The third stage (absent in intact rats) was characterized by slow waves and spindles of low amplitude in the cortex and low frequency theta rhythm, and it was considered as "drowsiness." Waking without theta rhythm, paradoxical sleep, and its forerunner intermediate stage were never found. Paroxystic-like EEG episodes were frequently observed. Thus, although present, the sleep-waking cycle is severely impaired in the caudopontine rats. The impairment is similar to that found previously in rats transected at the intercollicular or pretrigeminal level. The preparations were able to crawl abortively and to swallow liquid. Their respiratory rhythm was normal, but the heart rate increased. Thus, the caudal part of the preparations showed remarkable ability in controlling motor and vegetative functions.

The reactivity of the somesthetic S1 cortex during sleep and waking in the rat.

The reactivity of the somesthetic S1 cortex was studied in the rat, in the course of the seven principle stages of the sleep-waking cycle, in terms of the variation in amplitude of positive wave 4 of the evoked potential induced by stimulation of the thalamocortical radiations. The amplitude of positive wave 4 is minimal during waking with theta (attentive and/or active). It increases in the course of waking without theta, to reach its maximum during the stage of sleep with slow waves. The amplitude decreases with the deepening of slow sleep (spindles, intermediate stage) to a level near to waking without theta, during rapid sleep. No significant difference is observed during periods of eye movements bursts. The variability of the amplitude of intra-state responses is lowest in the phases of waking and paradoxical sleep. The recovery cycle of the S1 cortex responses is long (several hundreds of milliseconds). The rate of recovery is inversely proportional to the amplitude of the response to the conditioning stimulus. It is therefore higher during waking and rapid sleep than during the different stages of slow sleep. These results are integrated in the neurophysiological data of sleep in the rat.

Hippocampal theta activity in the acute precollicular rat.

In eleven acute precollicular rats, cortical and hippocampal EEG activity was recorded. Hippocampal theta activity of low frequency (mean 4.5 c/sec) was found in nine rats and in four occupied above 50% of the recording time. In contrast, in the cortex the synchronized EEG activity dominated. The electrical stimulation of the posterior hypothalamus induced the theta rhythm, or increased its frequency. The large amounts of theta activity in some rats suggests that the forebrain theta pacemakers, partly modulated by the posterior hypothalamus, are released from brain stem inhibitory influences.

Mutagen sensitivity and cancer susceptibility in patients with multiple primary cancers.

Thirty-two patients with multiple cancers were evaluated for in vitro sensitivity to mutagens and were compared with normal controls, Mutagen sensitivity was evaluated by exposing lymphocytes to mitomycin-C in vitro and estimating the mitomycin-induced chromosomal gaps and breaks (G/B) and sister chromatid exchanges (SCE) per cell. The results show significant differences between control and patient groups when spontaneous and induced G/B and SCE were evaluated. In addition, establishing the limit of normality for G/B and SCE, among the control group, the examined patients show largely positive levels in respect to both of the parameters used. In this study we discuss the usefulness of mutagen sensitivity as an indirect measure of DNA repair and genetic susceptibility to multiple primary cancers.

Aneusomy and tumor heterogeneity in metastatic breast cancer detected by fluorescence in situ hybridization.

Direct interphase cytogenetic analysis was performed on nuclei of metastatic effusions from breast cancer using fluorescence ill situ hybridization (FISH). DNA probes specific for repetitive pericentromeric regions on chromosomes 6, 8, 17, and human midi-satellite probe specific for one locus on the short arm of chromosome 1 were used to determine chromosome copy numbers in interphase tumor cells. The analysis showed cytogenetic heterogeneity in most nuclei examined with multiple subpopulations and a range of 0-6 chromosome signals per cell. The presence of subclones in these fluids, their correlation with highly malignant phenotypes and the diagnostic and prognostic applications of this method is discussed.