Plasmodium falciparum parasitized red blood cells modulate the production of endothelin-1 by human endothelial cells.

AIM: Plasmodium falciparum (P. falciparum) malaria is the most important parasitic infection of humans, responsible for about 2,000,000 deaths every year. Cytoadherence of P. falciparum parasitized erythrocytes (pRBC) to vascular endothelium contributes to the pathogenesis of severe malaria causing microcirculatory obstruction and subsequent tissue hypoxia. Several cytokines and vasoactive mediators are involved in this process. The aim of this paper was to investigate the production of endothelin-1 (ET-1), a potent vasoconstrictor agent, by endothelial cells from large vessels (human umbilical vein endothelial cells, HUVEC) or the microvasculature (human microvascular endothelial cells, HMEC-1), co-cultured with different strains of P. falciparum pRBC under normoxic or hypoxic conditions. METHODS: HMEC-1, immortalized by SV 40 large Tontigen, were maintained in MCDB 131 medium supplement ed with 10% fetal calf serum, 10 ng/ml of epidermal growth factor, 1 microg/ml of hydrocortisone, 2 mM glutamine, 100 U/ml of penicillin, 100 microg/ml of streptomycin and 20 mM Hepes buffer. The levels of ET-1 in the supernatants were measured by immunoenzymatic assay. RESULTS: The results indicated that IL1-beta and hypoxia were able to induce ET-1 production by both HUVEC and HMEC-1. However, the co-incubation of HUVEC or HMEC-1 with pRBC induced a dose-dependent decrease of both constitutive and IL1- or hypoxia-induced ET-1 production. The inhibition was independent from the parasite strain used and from the origin of endothelial cells. CONCLUSION: These results show that pRBC by modulating both constitutive and stimulated ET-1 release from endothelial cells can induce modifications of the vascular tone in different anatomical districts. This could be of relevance in the pathogenesis of severe malaria.

Single-cell analysis of cytokine production shows different immune profiles in multiple sclerosis patients with active or quiescent disease.

Peripheral blood mononuclear cells of multiple sclerosis (MS) patients were stimulated with myelin basic protein (MBP) together with anti-CD28 monoclonal antibody and staphylococcal enterotoxin B to optimize cytokine production by antigen-specific cells. Type 1 (IL-2, IL-12, IFNgamma) and pro-inflammatory (TNFalpha, IL-1beta, IL-6) cytokines were augmented in CD4+, CD8+, and CD14+ cells of acute MS patients and of patients undergoing disease reactivation. These cytokines were reduced in IFNbeta-treated and in stable MS patients; type 2 cytokines (IL-4, IL-10) were increased in these patients. Similar immune profiles are seen in MS patients in whom remission is naturally or pharmacologically (IFNbeta) achieved. Cytokine alterations are particularly evident in CD14+ cells, underlying their critical role in the modulation of the immune response.

Mucosal and systemic immune activation is present in human immunodeficiency virus-exposed seronegative women.

Immune parameters were analyzed in peripheral blood mononuclear cells (PBMC) and cervical mucosa biopsy specimens of human immunodeficiency virus (HIV)-seronegative women sexually exposed to HIV (exposed seronegative [ESN]), HIV-infected women, and healthy women without HIV exposure. HIV was not detected in PBMC or cervical mucosa biopsy specimens of ESN women. However, interleukin (IL)-6, IL-10, IL-12, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha and -beta mRNA were elevated in PBMC and cervical mucosa biopsy specimens of ESN and HIV-infected women; CCR5 and CXCR4 mRNA were augmented in cervical mucosa biopsy specimens, but not in PBMC, of ESN and HIV-infected women; HIV-specific IFN-gamma-secreting cells were detected in vaginal washes of ESN and HIV-infected women; and phenotypic alterations were present in PBMC of ESN women. These results suggest that active HIV infection is not required for T cell activation; immune alterations occur in women in whom HIV infection cannot be detected virologically or clinically.

A polymorphism in the repetitive (TGGA)n sequence 5' to the human myelin basic protein gene in Italian multiple sclerosis patients.

Human myelin basic protein (hMBP) gene is one of the candidate genes in the complex mosaic of multiple sclerosis (MS) susceptibility. In this study we verified the distribution of the polymorphism of the region 5' flanking the first exon of the hMBP gene, in 97 relapsing remitting, 74 primary progressive Italian MS patients, and in 236 healthy controls, using polymerase chain reaction (PCR) and gel electrophoresis analysis in this region from 1116 - 1540 nt. Three different band patterns were observed: one homozygote with a 354 bp long fragment, one homozygote with 424 bp long fragment and one heterozygote with both bands present. The short fragment was statistically more frequent in RRMS patients than in HC (P<0.05). The long fragment was more present in HC. Similarly the short homozygous pattern (354 bp/354 bp) was significantly higher in the RRMS patients versus the healthy controls (P<0.01). The sequence analysis of the hMBP alleles showed that while the long fragments matched the prototype sequence completely, all the short fragments showed a deletion of 70 bp from nt 1177 to nt 1247, which explains the short 354 bp allele detected by PCR. Moreover two single mismatches in positions 1386 (T-->C) and 1431 (G-->A), were present only in the short hMBP fragment.

Serum auto antibodies presence in multiple sclerosis patients treated with beta-interferon 1a and 1b.

To verify the possible effect of IFN-beta treatment on auto antibodies development in multiple sclerosis (MS) we studied 69 MS patients before and during the treatment with IFN-beta 1b (n=35) and IFN-beta 1a (n=20) for 27 and 12 months respectively, and, as controls, 14 untreated MS patients. The serum, collected every 3 months from all the patients, was investigated for the presence of antinuclear (ANA), anti-smooth muscle (ASMA), anti-mitochondrial (AMA), anti-native DNA (nDNA) anti-cardiolipin (aCL), anti-parietal cells (APCA), anti-microsomal (AMC) and anti-tireoglobulin (ATG) antibodies. Among the IFN-beta 1b-treated MS patients an increase of the frequency and of the level of ANA, AMC and ATG was observed. ASMA and ANA antibodies were already present in about 45% of the MS patients before the treatment and fluctuated over the time. In one patient the treatment was interrupted after 6 months because of the occurrence of high ASMA level and of an autoimmune hepatitis. The data obtained in the smaller number of MS patients treated with IFN-beta 1a were very similar. No increase in aCL level was observed during both the IFN treatments. Our results indicate that the treatment with IFN-beta induces an increase of AMC and ATG antibodies in MS patients and confirm that, although rare, autoimmune diseases could be observed. The possible effect of these auto antibodies on the treatment efficacy and on MS clinical course need to be further investigated.

Endothelin and nitric oxide levels in cerebrospinal fluid of patients with multiple sclerosis.

In order to investigate the potential role of endothelins (ETs) and nitric oxide (NO) in the pathogenesis of multiple sclerosis (MS) we evaluated the levels of these vasoactive mediators in cerebrospinal fluid (CSF) of relapsing remitting MS patients and in a group of subjects with other neurological diseases (OND) and in a control group of subjects without neurological disease. Eighty patients affected from clinically diagnosed MS were selected, 44 of them were studied during an acute clinical attack and 36 in a stable phase. The OND group included 21 subjects affected by degenerative non inflammatory (n=9) and inflammatory (n=12) neurological disease while the control group included 22 subjects with cancer of the prostate (n=11) and with bladder disease (n=11). ET levels were significantly increased in CSF of relapsing remitting MS patients with an acute clinical attack in comparison with those in a stable phase, the OND group and the control group. Moreover significant differences were observed among the four groups with regard to the NO levels: MS patients in a stable and acute phase like OND group have high levels of NO compared to the control group. Since the blood-brain barrier index values did not differ significantly between the three groups, the data of this study suggest an important role for NO and ET in cerebral microcirculation in MS patients.

Molecular evidences for a role of HSV-1 in multiple sclerosis clinical acute attack.

To verify the possible role of human herpesviruses as triggering or aggravating factors in relapsing-remitting multiple sclerosis (RRMS) clinical acute attack, we studied the prevalence of some herpesviruses in the peripheral blood mononuclear cells (PBMCs) collected from 22 MS patients during an MS relapse and in a stable phase and from 18 healthy controls (HC). DNA belonging to Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), Human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and Human Herpes virus 6 (HHV-6) has been searched by specific nested polymerase chain reaction (n-PCR). EBV and HHV6 DNA has been detected with high frequency in acute and stable MS and in healthy controls without significant differences. HCMV DNA was observed both in acute and stable MS but not in HC, and, more interestingly, HSV-1 DNA was only found in 13% of acute MS, while both stable MS and healthy controls were negative. On the basis of these results we focused on HSV-1, and to confirm them and to demonstrate that HSV-1 is actively replicating in MS patients during clinical relapse, we searched both messenger RNA (mRNA) and DNA of HSV-1 in the PBMCs of 15 acute MS patients and 15 healthy controls. We found HSV-1 mRNA and DNA in a significant number of acute MS patients but not in the control group. On the whole these data indicate that HSV-1 reactivate in the peripheral blood of MS patients during clinical acute attack and probably play a role in the triggering of MS relapses.

Serum eosinophil cationic protein (sECP) in subjects with a history of asthma symptoms with or without rhinitis.

BACKGROUND: Serum eosinophil cationic protein (sECP) has been proposed as a marker of disease activity in bronchial asthma. The study aimed to evaluate the role of sECP in screening asthmatics in a group of subjects with asthma and rhinitis symptoms, and the relationship between sECP and clinical and functional parameters of asthma. METHODS: A total of 185 subjects with asthma symptoms, 149 of them with rhinitis as well, underwent skin tests, spirometry, methacholine (MCH) test, blood sampling for eosinophil percentage (bEOS%) and sECP determination, and nasal secretions smear for eosinophil percentage (nEOS%) determination; PEF values, symptoms, and medication over a period of 4 weeks after sampling for sECP quantitation were recorded on a diary. RESULTS: A total of 99 (53%) subjects received a diagnosis of asthma (asthmatics), and 86 did not (nonasthmatics). In asthmatics, neither sECP nor bEOS% was significantly different from nonasthmatics. In asthmatics, sECP was higher in subjects with increased than in those with normal daily PEF variability (16.4, 6.8-24.4 vs 5.3, 3.9-8.4 microg/l; P<0.001). sECP was higher in moderate persistent asthma than in intermittent asthma (24.8, 10.6-53 vs 8.4, 5.6-14.1; P<0.05). In nonasthmatics (73 with a history of rhinitis), both sECP and bEOS% correlated with nEOS% (rho=0.35; P<0.01 and rho=0.53; P<0.001). CONCLUSIONS: In adults with asthma symptoms with or without rhinitis, sECP did not distinguish asthmatics from nonasthmatics. In asthmatics, sECP was associated with PEF variability and symptom severity. In subjects with asthma and rhinitis, as well as in subjects with only rhinitis, sECP levels are possibly influenced by nasal inflammation.

Different endothelins stimulate cytokine production by peritoneal macrophages and microglial cell line.

Endothelins (ETs), potent vasoconstricting peptides, are produced by macrophages upon stimulation and may participate in the amplification or regulation of the inflammatory response. However, it is not clear whether ETs can act in an autocrine manner on macrophages and which role they play in relationship with other cytokines. To address these issues, we studied the effects of ETs on the production of inflammatory cytokines by mouse peritoneal macrophages or by a retrovirus-transformed microglial cell line. Here, we report that ET-2, but not ET-1 or ET-3, is able to stimulate the production of interleukin-1 (IL-1) and interleukin-6 (IL-6) by peptone-elicited mouse macrophages (pMO). In contrast, ET-3 and ET-1, but not ET-2, are active on microglial cells. No tumour necrosis factor-alpha (TNF-alpha) or nitric oxide (NO) were detected in the supernatants of ET-stimulated cultures. The activity of ET-2 on pMO was time and dose dependent and was inhibited by the addition of ETA and ETB receptor antagonists, BQ123 and IRL1038, respectively. In addition, when pMO were stimulated by interferon-gamma (IFN-gamma) in the presence of ET-2, a significant inhibition of IL-6 and IL-1 production was observed compared with the effects of the same doses of IFN-gamma or ET-2 used separately. The inhibition was specifically due to the activity of ET-2, since it was reversed by the addition of BQ123 or IRL1038. Similar results were seen when the content of NO in the supernatants of pMO stimulated by IFN-gamma plus ET-2 was evaluated. These results suggest that ETs may possess both a pro-inflammatory action on macrophages from different tissues and a regulatory activity on IFN-gamma.

A rapid evaluation of phagocytosis and killing of Candida albicans by CD13+ leukocytes.

Flow cytometry can be adopted for routine monitoring of the immune functions of human polymorphonuclear leukocytes (PMNs) in several disease states. We recently developed a rapid and reproducible assay for the evaluation of the phagocytosis and killing of Candida albicans blastospores by human PMNs. Whole blood leukocytes were incubated with opsonized fluorescein isothiocyanate-labeled (FITC-labeled) blastospores for phagocytosis and killing assays. To discriminate between ingested, membrane-bound and free C. albicans blastospores, ethidium bromide (EtBr) was added to the samples prior to the flow cytometric analysis. EtBr induces a loss of green fluorescence in non-phagocytized C. albicans blastospores. Phagocytosis is determined by gating the phagocytes and calculating the percentage of phagocyte-associated green fluorescent cells. Intracellular killing is determined by first lysing phagocytes by hypotonic shock and then adding propidium iodide (PI) in order to identify red dead blastospores. Killing is measured in terms of the percentage of double-marked blastospore cells. We suggest that this method is a reliable and inexpensive technique to evaluate the immune reactivity of PMNs and peripheral blood monocytes (PBMs) in cases of immunosuppression.

Evidence for a missed signal to the CD8+ cells in CSF of multiple sclerosis patients.

Peripheral blood (PB) and cerebrospinal fluid (CSF) lymphocyte subpopulations, defined by various T-cell specific monoclonal antibodies and flow cytometry, were analysed in 44 relapsing remitting multiple sclerosis (RRMS) patients (including 21 subjects in the acute phase and 23 in the stable phase), 40 chronic-progressive multiple sclerosis (CPMS) patients, and 24 patients with other neurological diseases (OND), in order to verify the presence of any abnormality in the lymphocyte subset pattern. A significant increase in the total number of T-lymphocytes and the CD4+ subpopulation was found in the PB of the MS patients in comparison with the OND group. Moreover, a not statistically significant increase in CD4+ cells was observed in the CSF of MS patients. A statistically significant increase was also found in the CD4+ Leu 8+ (suppressor inducer) cells in the CSF of all of the MS groups. Finally, the CD8+ (suppressor/cytotoxic) cell levels, were significantly lower in the CSF of CPMS and stable RMS patients than in the CSF of the OND patients. As a whole, our data suggest that the immunosuppressive deficit that seems to be a constant finding in MS is not due to a decrease in suppressor inducer cell levels, as previously suggested, but may be caused by a missed or altered signal from the suppressor inducer to CD8+ suppressor cells.

Binding sites in fibronectin for an enterotoxigenic strain of E. coli B342289c.

The binding of fibronectin and fibronectin fragments to the enterotoxigenic strain E. coli B34289c was studied. E. coli cells bound to two distinct sites of fibronectin, one being the N-terminal domain, which also contains the binding sites for staphylococci and streptococci, and the other located within the central heparin binding region. In addition, the N-terminal and the heparin binding domain mediated the attachment of bacteria in a solid phase binding assay. E. coli cells expressed two classes of receptors, the first, a 17 kDa protein, recognized by the N-terminal fragment and the second, having a mol. mass of 55 kDa, which interacts with the internal heparin binding domain. Bacterial receptors, which bind the N-terminal end of fibronectin, may be structurally related.