The interleukin-1ß/CXCL1/2/neutrophil axis mediates host protection against group B streptococcal infection.

Previous studies have indicated that group B streptococcus (GBS), a frequent human pathogen, potently induces the release of interleukin-1ß (IL-1ß), an important mediator of inflammatory responses. Since little is known about the role of this cytokine in GBS disease, we analyzed the outcome of infection in IL-1ß-deficient mice. These animals were markedly sensitive to GBS infection, with most of them dying under challenge conditions that caused no deaths in wild-type control mice. Lethality was due to the inability of the IL-1ß-deficient mice to control local GBS replication and dissemination to target organs, such as the brain and the kidneys. Moreover, in a model of inflammation induced by the intraperitoneal injection of killed GBS, a lack of IL-1ß was associated with selective impairment in the production of the neutrophil chemokines CXCL1 and CXCL2 and in neutrophil recruitment to the peritoneal cavity. Decreased blood neutrophil counts and impaired neutrophil recruitment to the brain and kidneys were also observed during GBS infection in IL-1ß-deficient mice concomitantly with a reduction in CXCL1 and CXCL2 tissue levels. Notably, the hypersusceptibility to GBS infection observed in the immune-deficient animals was recapitulated by neutrophil depletion with anti-Gr1 antibodies. Collectively, our data identify a cytokine circuit that involves IL-1ß-induced production of CXCL1 and CXCL2 and leads the recruitment of neutrophils to GBS infection sites. Moreover, our data point to an essential role of these cells in controlling the progression and outcome of GBS disease.

Class II-specific histone deacetylase inhibitors MC1568 and MC1575 suppress IL-8 expression in human melanoma cells.

Here, we explored the effects of the novel class II-specific histone deacetylase inhibitors (HDACis) MC1568 and MC1575 on interleukin-8 (IL-8) expression and cell proliferation in cutaneous melanoma cell line GR-M and uveal melanoma cell line OCM-3 upon stimulation with phorbol 12-myristate 13-acetate (PMA). We found that PMA upregulated IL-8 transcription via the AP-1 binding site and identified c-Jun as the transcription factor involved in this eventS. MC1568 and MC1575 inhibited IL-8 levels and cell proliferation in either unstimulated or PMA-stimulated melanoma cells. They acted by suppressing (i) c-Jun binding to the IL-8 promoter, (ii) recruitment of histones 3 and 4, RNA polymerase II and TFIIB to the c-Jun promoter, and (iii) c-Jun expression. Our findings provide new insights into mechanisms underlying anti-tumoral activities of class II-specific HDACis in human melanoma and suggest that they may constitute a novel therapeutic strategy for improving the treatment of this cancer.

FOXE1 is a target for aberrant methylation in cutaneous squamous cell carcinoma.

BACKGROUND: Several cancer-related genes are silenced by promoter hypermethylation in skin cancers. However, to date the somatic epigenetic events that occur in cutaneous squamous cell carcinoma (SCC) tumorigenesis have not been well defined. OBJECTIVES: To examine epigenetic abnormalities of FOXE1, a gene located on chromosome 9q22, a region frequently lost in SCC. METHODS: We investigated the methylation status of FOXE1 in 60 cases of cutaneous SCC by methylation-specific polymerase chain reaction, and comparatively examined mRNA and protein expression by real-time polymerase chain reaction and Western blot, respectively. RESULTS: We found a higher frequency of FOXE1 promoter hypermethylation in SCCs (55%), as compared with the adjacent uninvolved skin (12%) and blood control samples (9.5%). FOXE1 methylation was frequently seen in association with a complete absence of or downregulated gene expression. Treatment with the demethylating agent 5-Aza-2'-deoxycytidine resulted in profound reactivation of FOXE1 expression. CONCLUSIONS: These results indicate that FOXE1 is a crucial player in development of cutaneous SCC.

Investigation into FOXE1 genetic variations in cutaneous squamous cell carcinoma.

BACKGROUND: FOXE1 is a candidate tumour suppressor gene at human chromosome locus 9q22. This is a region frequently lost in squamous cell cancer. OBJECTIVES: To assess the influence of FOXE1 variations on genetic susceptibility to cutaneous squamous cell carcinoma (SCC). METHODS: We performed mutational analysis of FOXE1 in 320 DNA samples isolated from 60 SCC specimens, 60 adjacent histologically normal skin samples and 200 blood samples. RESULTS: No somatic mutations were evident. Instead the polyalanine tract showed marked variation in its length between samples from patients with SCC and normal controls. CONCLUSIONS: These results imply that another tumour suppressor gene at this locus may be more important than FOXE1 in skin carcinogenesis and suggest that variation in the FOXE1 polyalanine tract length predisposes to cutaneous SCC.

Vitreous polyamines spermidine, putrescine, and spermine in human proliferative disorders of the retina.

BACKGROUND/AIMS: Many cytokines are involved in the pathogenesis of retinal proliferative diseases, but none has been shown to be related to a specific disorder. The aim of this study was to provide a selective marker of diabetes induced proliferative retinopathies. METHODS: 10 vitreous samples from 10 subjects affected by quiescent proliferative diabetic retinopathy (PDR), 20 vitreous samples from 20 subjects affected by active PDR, and 15 samples from 15 patients with proliferative vitreoretinopathy (PVR) were studied. Samples from 18 patients with a macular hole (n = 8) or pucker (n = 10) served as controls. Vitreous samples were obtained via pars plana vitrectomy. The polyamines spermidine, putrescine, and spermine, vascular endothelial growth factor (VEGF), interleukin 8 (IL-8), and transforming growth factor 1beta (TGF-1beta) were measured by high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA), and the correlation coefficients between the vitreous polyamine content and VEGF, IL-8, and TGF-1beta levels were determined. RESULTS: Spermidine and putrescine were expressed in normal vitreous, but spermine was not detectable. In all the test groups spermidine was 3-4 times higher than in control vitreous and putrescine was similarly lower. The spermine content was up to 15 times higher only in vitreous from patients affected by PDR. Correlation coefficients showed that the spermidine and putrescine level variations correlated with the VEGF and IL-8 content in the active PDR and PVR groups, but not in those with quiescent PDR patients, while spermine was correlated to these cytokines in PDR, but not in PVR groups. CONCLUSIONS: These data suggest a significant role for spermidine and putrescine as markers of proliferative diseases of the retina. The increase in spermine, restricted to diabetic states, may indicate that this polyamine is a unique and specific index of PDR.

Anion transport in normal erythrocytes, sickle red cells, and ghosts in relation to hemoglobins and magnesium.

"Band 3," an integral membrane protein of red blood cells, plays a relevant role in anionic transport. The C- and N-terminal portions of band 3 are cytoplasmatics, and the last is the link site for different glycolitic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, and hemoglobin. All or some of these interactions on the CDB3 protein could allow a subtle modulation of anion flux. The interaction among HbA, Mg(2+), and membrane proteins has been sufficiently investigated, but not the effect of Mg(2+) on pathological hemoglobin in relation to the influx of the SO(4)(2-). The aim of this study was to evaluate the involvement of hemoglobin S in sulfate transport. This has been measured with native and increased concentrations of Mg(2+), using normal erythrocytes containing HbA, sickle red cells containing HbS, or ghosts obtained from both erythrocytes and normal erythrocytes ghosts with HbS added. The magnitude of the SO(4)(2-) rate constant measured in normal red blood cells increased markedly when measured in the presence of varied Mg(2+) concentrations. The results show that a low increase of intracellular Mg(2+) concentrations exercises a different HbA modulation on band 3 protein and consequently higher anion transport activity. The same experiments carried out in sickle red cells showed that the SO(4)(2-) rate constant measured in the presence of native concentrations of Mg(2+) was normal, compared to normal red cells, and was not affected by any increase of intracellular Mg(2+). Our suppositions with regard to the importance exercised by the hemoglobin and the Mg(2+) on the SO(4)(2-) influx were confirmed by comparison of the data obtained through measuring SO(4)(2-) influx with native and increased concentrations of Mg(2+) in both normal and sickle red cell ghosts. Both revealed the same sensitivity to Mg(2+) due to withdrawal of hemoglobins. The incorporation of HbS in normal as well as in sickle red cell ghosts reduced the Mg(2+) response to sulfate influx in both the reconstituted ghosts. Our research demonstrated that the different effects exercised on the rate constants of SO(4)(2-) influx in normal (HbA) and sickle red cells (HbS) by the increased intracellular Mg(2+) could be ascribed to the physical-chemical influence exercised either on the hemoglobins or on the intracellular contents of erythrocytes.

Prostaglandin E2 signalling pathway in human T lymphocytes from healthy and conjunctiva basal cell carcinoma-bearing subjects.

Prostaglandin E-induced signal transduction pathways in human T cells from healthy and uveal melanoma-bearing subjects were studied. Transfection experiments showed that PGE2 was able to phosphorylate and activate the fusion trans-activator of the cAMP responsive element-binding protein (CREB). Phosphorylation was at least partially mediated by protein kinase A, as evidenced by the effects of specific kinase inhibitors. Western blotting experiments, which were performed to identify the CREB/ATF2 family members involved in the response to PGE2, revealed a modulation of proteins CREB1, CREB2 and ATF2 and phosphorylation of the 43 kDa form of CREB. Experiments of immunoprecipitation with CREB-binding protein (CBP) demonstrated that, after PGE2 treatment, all of the CREB/ATF isoforms studied, as well as the phosphorylated form of CREB (p-CREB), interacted with CBP. In basal conditions, T cells from patients with conjunctiva basal cell carcinoma showed the presence of p-CREB, which coimmunoprecipitated with CBP. CREB phosphorylation did not modify after PGE2 treatment whereas the p-CREB fraction bound to CBP increased in a delayed manner compared to normal subjects.

Modified responses to PGE2 in polyamine biosynthesis by T lymphocytes of gastric- and conjunctiva basal cell-carcinoma patients.

The response to Prostaglandin (PG) E2 of T cells from gastric carcinoma (GC)- and conjunctiva-basal cell carcinoma (conjunctiva-BCC)-bearing patients has been studied in relation to polyamine metabolism. Polyamines are crucial co-factors in cell growth as well as differentiation and many works report that lymphocyte spermine (SP), spermidine (SPD) and putrescine (PUT) levels may be related to tumor proliferation. The present work aims to detect the basal and PGE2 induced concentrations of these polyamines and cAMP, ornithine decarboxylase (ODC), spermine N1-acetyltransferase (SAT) activities of T lymphocytes drawn from patients suffering from GC and conjunctiva-BCC since many carcinomas are characterized by high levels of PGE2. Data obtained from lymphocytes of neoplastic subjects were compared with those derived from PGE2-treated control lymphocytes. Results highlight a very significant increase of all the polyamine metabolites in PGE2-treated T cells from neoplastic patients in respect to the untreated and PGE2-treated control lymphocytes. Therefore, it is conceivable that the PGE2 content increase, often occurring during the epithelial tumour development, may contribute, through enhancement of polyamine metabolism, to tumor progression.

Effects of vitamin E and prostaglandin E2 on expression of CREB1 and CREB2 proteins by human T lymphocytes.

Both prostaglandins (PGs) and vitamin E are known to deeply affect immune responses. It is shown here that they both influence T cell-mediated immunity through reciprocal interference on the expression of cyclic-AMP responsive element binding (CREB) family proteins. CREB1 protein of human T lymphocytes was significantly modulated by a brief treatment of 5 to 10 min with PGE2. On the contrary, vitamin E appeared to be ineffective on the CREB1 behavior, while it abolished the PGE2-induced modulation of this protein. The CREB2 protein expression was also affected by PGE2 treatment, but a longer period of incubation (>20 min) was needed to observe these changes. Vitamin E showed a strong enhancing effect on CREB2 that was partially reversed by the subsequent treatment with PGE2. Our results support the idea that there is reciprocal interference between PGE2 and vitamin E on PGE2-induced signals in T lymphocytes. These data are in agreement with the reports concerning different cell systems and experimental conditions.

Glycolytic enzymes in polyamine-treated bovine retina.

The retina is characterized by glycolysis under aerobic conditions, mediated by lactate dehydrogenase isoenzyme-5 (LDH-5) as well as by the soluble isoenzyme of malate dehydrogenase. Bovine retina LDH and MDH isoenzymes and their activities were studied after polyamine treatment. Our results showed that LDH-5 isoenzyme presented the highest activity in untreated as well as in putrescine-treated retina. Decreased activity was present when the retina was treated with spermidine or spermine. It was demonstrated that retinic LDH-5 had a high affinity for lactate which enabled the isoenzyme to be more effective than the other LDH isoenzymes in the conversion of NADH to NAD. Therefore, the putrescine enhancing LDH-5 activity appeared to be capable of stimulating NAD-mediated rhodopsin regeneration. Putrescine induced a marked increase of both MDH isoenzymes--soluble (s-MDH) and mitochondrial (m-MDH), while spermine and spermidine mostly affected the soluble form of the enzyme. Putrescine induced a three-fold increase in s-MDH and m-MDH activities, while spermine and spermidine induced a four to five-fold increase in s-MDH. These results document the differential effects of polyamine treatment on LDH and MDH isoenzyme activities.

Polyamine metabolism in prostaglandin E2-treated human T lymphocytes.

The effects of Prostaglandin (PG) E2 treatment of human T lymphocytes on polyamine metabolism were investigated. PGE2 is known to inhibit lymphocyte proliferation, while polyamines play an important role in several biochemical processes leading to increased cell growth. Preincubation of T lymphocytes with PGE2 (10(-6) M) for 10 min was able to increase ornithine decarboxylase (ODC) activity and putrescine as well as spermine levels, while spermidine concentration was drastically reduced. After 30 and 60 min of treatment, a decrease in ODC activity and putrescine concentration was observed. On the contrary, the initial inhibition of spermine-N1-acetyltransferase (SAT) activity was followed by a progressive increase of this catabolic enzyme. These changes were related to modifications of cAMP concentrations. Our data may help clarify the mechanisms underlying the biphasic effect of PGE2, which ultimately leads to inhibition of cell proliferation.

Resistance to PGE2 inhibition of PWM-stimulated lymphocytes from neoplastic patients.

PGE2 treatment of mononuclear cells from patients with different types of neoplasias was unable to decrease either the number of plaque-forming cells or the expression of CD71 and CD25 in PWM-driven cultures. In contrast, in previous studies, PGE2 inhibited these parameters in cultured mononuclear cells from normal volunteers. Surgical treatment of cancer patients did not modify the lymphocyte sensitivity to PGE2 after 1 week, but at 2 and 6 months after therapeutical treatment, the inhibition values of the parameters studied were almost similar or very similar to those of normal lymphocytes. The reduction of PGE2 sensitivity in cancer patients was related to the increase of PGE2 levels and, probably, to a PGE2 receptor saturation. A restoration of PGE2-induced inhibition some months after therapy could be due to the decrease in PGE2 levels and to receptor unsaturation.

Prostaglandin E2 induction of binding activity to CRE and AP-2 elements in human T lymphocytes.

Prostaglandins of the E series are immunomodulatory agents which exert inhibitory as well as stimulatory effects on a variety of immune responses. Since it is known that PGE2 is able to increase cAMP levels, we investigated whether it can affect gene expression through the activation of the transcription factors which bind enhancer elements in the promoter regions of cAMP-regulated genes. Using electrophoretic mobility shift assay, we demonstrated that a short treatment of human T lymphocytes with PGE2 induces specific binding activity to CRE and AP-2, but not AP-1, DNA elements. Since the okadaic acid, a potent protein phosphatase inhibitor, prolongs the induction of the binding activity, phosphorylation events are likely to occur. This activity seems to be due to increased cAMP levels because forskolin and IBMX mimic the effects of PGE2. More interestingly, transfection experiments with CRE-CAT plasmide show that PGE2 activates the transcription of a CRE-containing promoter. These data support the positive role for PGE2 on some immune functions.

PGE2-induced immunoregulation mediated by cytokine production from cultures of human peripheral T lymphocytes.

Prostaglandins (PGs) are generally known to exert inhibitory as well as some enhancing effects on the immune system. This study was performed to assess the influence of the exogenous PGs and cyclic adenosine monophosphate (cAMP)-elevating agents on cytokine production by PWM-stimulated human T lymphocytes. Peripheral blood T lymphocytes from healthy donors were pretreated for 30 min at 25 degrees C with PGE2 (10(-4) to 10(-7) M) PGF1 alpha, IBMX or Forskolin (10(-4) to 10(-5) M) and cultured for 7 days in the presence of Pokeweed Mitogen (PWM), PGE2, IBMX and Forskolin, but not PGF1 alpha, significantly increased IL-6 production while inhibited IL-2, IL-3, IL-4, IFN-gamma, TNF-alpha and GM-CSF production. Our data indicate that cAMP-elevating agents can profoundly affect cytokine secretion by T cells in PWM-driven cultures and that this effect is dose-dependent. The results reported here are compatible with the existence of separate pathways of gene induction for IL-2, IL-3, IL-4, IFN-gamma, TNF-alpha and GM-CSF on the one hand and IL-6 on the other one.

Prostaglandins affect the respiratory burst of human neutrophils.

The effects of prostaglandins on superoxide generation by neutrophils were investigated, since these arachidonic acid metabolites are both involved in the early phase of the inflammatory process and during later stages of neutrophil function. Preincubation of these cells for five minutes with concentrations of PGE2 ranging from 10(-7) to 10(-4) M was able to significantly reduce superoxide production in PMA-stimulated neutrophils. Other pro-inflammatory PGs tested, such as PGE1, PGF1 alpha, PGF2 alpha, inhibited the respiratory burst. The PGE2-induced inhibition was compared to that exerted by staurosporine, a PKC inhibitor. The effects of the two drugs were not additive, since the combinations of PGE2 and staurosporine reduced O2- production to the same extent as staurosporine alone. Possible interferences between PKA- and PKC-mediated transduction signals are discussed.

[Not Available]. / Malattia acuta esantematica da parvovirus umano.

In June 1994 have been seen 16 patients with HPV B19 infection. The diagnosis was based on a positive E.L.I.S.A. test for IgM antibodies against HPV B19. No one of the patients had hematologic manifestations. Only one patient had fever, arthralgia and headache without skin involvement. The other 15 patients had skin lesion: rubella like rush in 11 cases, haemorragic exanthema in 2 and erythema infectious in 2 cases. Two children had liver involvement and one kidney involvement. The authors of this study remark the broad spectrum of clinical manifestations of HBV B19 infection and suppose a large diffusion of this disease, even in the symptomatic form.