Virulent infections due to alpha-haemolytic streptococci in cancer patients and their management

Streptococci of the viridans group have long been considered to be minor pathogens, except in bacterial endocarditis. For some years, however, these microorganisms have been the cause of serious bacteraemia in neutropenic patients receiving intensive chemotherapy. These infections can lead to severe complications such as endocarditis, respiratory distress syndromes or shock, and are associated with a mortality rate ranging from 6-30%. The principal risk factors for these infections are profound neutropenia, antibiotic prophylaxis with quinolones or cotrimoxazole, large doses of cytosine arabinoside, a recent history of chemotherapy, oropharyngeal mucositis and viridans streptococcal colonization. Protective factors are the early administration of parenteral antibiotics during periods of neutropenia, or the prophylactic administration of penicillin. Although the introduction of penicillin to prophylactic antibiotic regimens has led to a decrease in the incidence of these infections, the emergence of strains resistant to beta-lactams is a worrying problem which could compromise this type of treatment.

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.

Sex difference in hepatic peroxisome proliferator-activated receptor alpha expression: influence of pituitary and gonadal hormones.

Peroxisome proliferator-activated receptor (PPAR) alpha is a nuclear receptor that is mainly expressed in tissues with a high degree of fatty acid oxidation such as liver, heart, and skeletal muscle. Unsaturated fatty acids, their derivatives, and fibrates activate PPARalpha. Male rats are more responsive to fibrates than female rats. We therefore wanted to investigate if there is a sex difference in PPARalpha expression. Male rats had higher levels of hepatic PPARalpha mRNA and protein than female rats. Fasting increased hepatic PPARalpha mRNA levels to a similar degree in both sexes. Gonadectomy of male rats decreased PPARalpha mRNA expression to similar levels as in intact and gonadectomized female rats. Hypophysectomy increased hepatic PPARalpha mRNA and protein levels. The increase in PPARalpha mRNA after hypophysectomy was more pronounced in females than in males. GH treatment decreased PPARalpha mRNA and protein levels, but the sex-differentiated secretory pattern of GH does not determine the sex-differentiated expression of PPARalpha. The expression of PPARalpha mRNA in heart or soleus muscle was not influenced by gender, gonadectomy, hypophysectomy, or GH treatment. In summary, pituitary-dependent hormones specifically regulate hepatic PPARalpha expression. Sex hormones regulate the sex difference in hepatic PPARalpha levels, but not via the sexually dimorphic GH secretory pattern.

The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment.

The fasting-induced adipose factor (FIAF, ANGPTL4, PGAR, HFARP) was previously identified as a novel adipocytokine that was up-regulated by fasting, by peroxisome proliferator-activated receptor agonists, and by hypoxia. To further characterize FIAF, we studied regulation of FIAF mRNA and protein in liver and adipose cell lines as well as in human and mouse plasma. Expression of FIAF mRNA was up-regulated by peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARbeta/delta agonists in rat and human hepatoma cell lines and by PPARgamma and PPARbeta/delta agonists in mouse and human adipocytes. Transactivation, chromatin immunoprecipitation, and gel shift experiments identified a functional PPAR response element within intron 3 of the FIAF gene. At the protein level, in human and mouse blood plasma, FIAF was found to be present both as the native protein and in a truncated form. Differentiation of mouse 3T3-L1 adipocytes was associated with the production of truncated FIAF, whereas in human white adipose tissue and SGBS adipocytes, only native FIAF could be detected. Interestingly, truncated FIAF was produced by human liver. Treatment with fenofibrate, a potent PPARalpha agonist, markedly increased plasma levels of truncated FIAF, but not native FIAF, in humans. Levels of both truncated and native FIAF showed marked interindividual variation but were not associated with body mass index and were not influenced by prolonged semistarvation. Together, these data suggest that FIAF, similar to other adipocytokines such as adiponectin, may partially exert its function via a truncated form.

Effects of salbutamol on the contractile properties of human skeletal muscle before and after fatigue.

AIM: The study examined the effects of an oral acute administration of the beta2-agonist salbutamol (Sal) (6 mg) vs. placebo on muscle strength and fatigability in 12 non-asthmatic recreational male athletes in a randomized double-blind protocol. METHODS: Contractile properties of the right quadriceps muscle were measured during electrical stimulations, i.e. twitch, 1-s pulse trains at 20 (P(20) ) and 80 Hz (P(80) ) and during maximal voluntary isometric contraction (MVIC) before (PRE) and after (POST) a fatigue-producing protocol set by an electromyostimulation (30 contractions, frequency: 75 Hz, on-off ratio: 6.25-20s). In addition, the level of muscle voluntary activation was measured. RESULTS: In PRE and POST conditions, the peak torque (PT) of twitch, P(80) and MVIC were not modified by the treatment. The PT in POST P(20) was slightly, although not significantly, less affected by fatigue in Sal compared with placebo condition. Moreover, twitch half-relaxation time at PRE was smaller under Sal than under placebo (P < 0.05). No significant changes in the degree of voluntary activation were observed with Sal treatment in PRE or POST condition. CONCLUSION: Although these findings did not exclude completely an effect of Sal on peripheral factors of human skeletal muscle, oral acute administration of the beta2-agonist Sal seems to be without any relevant ergogenic effect on muscle contractility and fatigability in non-asthmatic recreational male athletes.

Revealing a subclinical salt-losing phenotype in heterozygous carriers of the novel S562P mutation in the alpha subunit of the epithelial sodium channel.

OBJECTIVE: Pseudohypoaldosteronism type I (PHA1) is a rare inborn disease causing severe salt loss. Mutations in the three coding genes of the epithelial sodium channel (ENaC) are responsible for the systemic autosomal recessive form. So far, no phenotype has been reported in heterozygous carriers. PATIENTS: A consanguineous family from Somalia giving birth to a neonate suffering from PHA1 was studied including clinical and hormonal characteristics of the family, mutational analysis of the SCNN1A, SCNN1B, SCNN1G and CFTR genes and in vitro analysis of the functional consequences of a mutant ENaC channel. RESULTS: CFTR mutations have been excluded. SCNN1A gene analysis revealed a novel homozygous c.1684T > C mutation resulting in a S562P substitution in the alphaENaC protein of the patient. Functional analysis showed a significantly reduced S562P channel function compared to ENaC wild type. Protein synthesis and channel subunit assembly were not altered by the S562P mutation. Co-expression of mutant and wild-type channels revealed a dominant negative effect. In heterozygote carriers, sweat sodium and chloride concentrations were increased without additional hormonal or clinical phenotypes. CONCLUSION: Hence, the novel mutation S562P is causing systemic PHA1 in the homozygous state. A thorough clinical investigation of the heterozygote SCNN1A mutation carriers revealed increased sweat sodium and chloride levels consistent with a dominant effect of the mutant S562P allele. Whether this subclinical phenotype is of any consequence for the otherwise asymptomatic heterozygous carriers has to be elucidated.

Relationships among endogenous ouabain, alpha-adducin polymorphisms and renal sodium handling in primary hypertension.

OBJECTIVE: The basolateral Na pump drives renotubular reabsorption. In cultured renal cells, mutant adducins, as well as sub-nanomolar ouabain concentrations, stimulate the Na-K pump. METHODS: To determine whether these factors interact and affect Na handling and blood pressure (BP) in vivo, we studied 155 untreated hypertensive patients subdivided on the basis of their plasma endogenous ouabain or alpha-adducin genotype (ADD1 Gly460Trp-rs4961). RESULTS: Under basal conditions, proximal tubular reabsorption and plasma Na were higher in patients with mutated Trp ADD1 or increased endogenous ouabain (P = 0.002 and 0.05, respectively). BPs were higher in the high plasma endogenous ouabain group (P = 0.001). Following volume loading, the increment in BP (7.73 vs. 4.81 mmHg) and the slopes of the relationship between BP and Na excretion were greater [0.017 +/- 0.002 vs. 0.009 +/- 0.003 mmHg/(muEq min)] in ADD1 Trp vs. ADD1 Gly carriers (P < 0.05). BP changes were similar, whereas the slopes of the relationship between BP and Na excretion were lower [0.016 +/- 0.003 vs. 0.008 +/- 0.002 mmHg/(muEq min)] in patients with low vs. high endogenous ouabain (P < 0.05). In patients with high endogenous ouabain, volume loading increased the BP in the ADD1 Trp group but not in the Gly group (P < 0.05). Thus, patients with ADD1 Trp alleles are sensitive to salt and tubular Na reabsorption remains elevated after volume expansion. CONCLUSION: With saline loading, BP changes are similar in high and low endogenous ouabain patients, whereas tubular Na reabsorption increases in the high endogenous ouabain group. Saline loading unmasks differences in renal Na handling in patients with mutant adducin or high endogenous ouabain and exposes an interaction of endogenous ouabain and Trp alleles on BP.

Toll-like receptor 3 expressed by melanoma cells as a target for therapy?

PURPOSE: The immunomodulatory properties of Toll-like receptors (TLR) agonists have inspired their use as experimental adjuvants for vaccination of cancer patients. However, it is now well recognized that TLR expression is not restricted to immune cells but can also be found in many cell types, including those giving rise to tumors. It is therefore mandatory to explore the potential effects of TLR triggering directly on tumor cells. EXPERIMENTAL DESIGN: In the present work, we have investigated TLR3 protein expression in melanoma cell lines derived from patients, and analyzed the effects of TLR3 agonists on tumor cell survival. Moreover, we used RNA interference to stably knock down TLR3 expression and study the involvement of this receptor in dsRNA-induced effects on melanoma cells viability. RESULTS: Human melanoma cells can express functional TLR3 protein. Interestingly, the engagement of the receptor by TLR3 agonists can directly inhibit cell proliferation and induce tumor cell death when combined to treatment with either type I IFN or protein synthesis inhibitors. These effects were shown by RNA interference to be largely dependent on TLR3. Moreover, TLR3-mediated cell death involves the activation of caspases and engages both extrinsic and intrinsic apoptotic pathways. CONCLUSION: TLR3 protein can be expressed in human melanoma cells, where it can deliver proapoptotic and antiproliferative signaling. Altogether, these results suggest that TLR3 agonists represent very promising adjuvants for cancer vaccines not only based on their well-described immunostimulatory properties, but also due to their newly identified cytostatic and cytotoxic effects directly on tumor cells.

In vivo and in vitro development of alpha-MSH and ACTH in the embryonic and postnatal rat brain.

The appearance of immunoreactive alpha-melanotropin (alpha-MSH) and adrenocorticotropin (ACTH) during development was studied in 3 areas of the rat brain--cerebral hemispheres, midbrain and hindbrain--from embryonic day (ED) 13-14 until day 21 postnatally. The alpha-MSH content in vivo was always highest in the midbrain; a peak content at birth was followed by a transient decline and a later, higher plateau from postnatal day 7 onwards. The alpha-MSH content in the cerebral hemispheres rose progressively after birth reaching a peak at day 21. Values in the hindbrain rose at day 3 and changed relatively sue taken at ED 15-16 showed a gradual increase in alpha-MSH content over the 20 days. The alpha-MSH content of hindbrain cultures remained at constant low levels, while no alpha-MSH was detectable in cerebral hemisphere cultures. ACTH appeared in vivo earlier than alpha-MSH and was detectable in embryonic brains at ED 13-14. A transient rise was seen at ED 17-18 and major peaks at birth, day 2 and day 3, in the midbrain, hemispheres and hindbrain, respectively. In vitro, the ACTH content increased in all brain regions during the first 5 days in culture and showed no further change thereafter. Comparisons of the in vivo and in vitro development of alpha-MSH and ACTH demonstrate that (i) these two peptide systems are independent in respect to their localization and time of appearance; (ii) they undergo maturation both in vivo and in vitro; (iii) epigenetic factors, such as interactions with other neurotransmitter systems may modulate the developmental pattern of these two peptides.

A comparison of alpha and gamma spectrometry for environmental natural radioactivity surveys.

An alpha-spectrometry, using automated borate fusion and sequential extraction and exchange chromatography, was used to determine the uranium and thorium based on environmental radioactivity of 20 soil samples. The same set of the samples was analysed using gamma-spectrometry with an HPGe detector. The two data sets were checked for coherence using Z-score and chi2 statistical tests. We show that gamma-spectrometry is a valid alternative to time-consuming alpha-spectrometry for the determination of natural uranium and thorium activity in soil (activity range: 12.5-58.2 Bq/kg). The measured activities were compared with the theoretical activities to ensure secular equilibrium in the 238U and 232Th series. For 226Ra, a special study was made on deconvolution of the 186 keV multiplet with the Levenberg-Marquardt algorithm. Finally, the combined use of Z-score and chi2-tests was found to be a powerful tool for comparing the results obtained with two different methods.

Antagonistic regulation of a proline-rich transcription factor by transforming growth factor beta and tumor necrosis factor alpha.

Transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha) often exhibit antagonistic actions on the regulation of various activities such as immune responses, cell growth, and gene expression. However, the molecular mechanisms involved in the mutually opposing effects of TGF-beta and TNF-alpha are unknown. Here, we report that binding sites for the transcription factor CTF/NF-I mediate antagonistic TGF-beta and TNF-alpha transcriptional regulation in NIH3T3 fibroblasts. TGF-beta induces the proline-rich transactivation domain of specific CTF/NF-I family members, such as CTF-1, whereas TNF-alpha represses both the uninduced as well as the TGF-beta-induced CTF-1 transcriptional activity. CTF-1 is thus the first transcription factor reported to be repressed by TNF-alpha. The previously identified TGF-beta-responsive domain in the proline-rich transcriptional activation sequence of CTF-1 mediates both transcriptional induction and repression by the two growth factors. Analysis of potential signal transduction intermediates does not support a role for known mediators of TNF-alpha action, such as arachidonic acid, in CTF-1 regulation. However, overexpression of oncogenic forms of the small GTPase Ras or of the Raf-1 kinase represses CTF-1 transcriptional activity, as does TNF-alpha. Furthermore, TNF-alpha is unable to repress CTF-1 activity in NIH3T3 cells overexpressing ras or raf, suggesting that TNF-alpha regulates CTF-1 by a Ras-Raf kinase-dependent pathway. Mutagenesis studies demonstrated that the CTF-1 TGF-beta-responsive domain is not the primary target of regulatory phosphorylations. Interestingly, however, the domain mediating TGF-beta and TNF-alpha antagonistic regulation overlapped precisely the previously identified histone H3 interaction domain of CTF-1. These results identify CTF-1 as a molecular target of mutually antagonistic TGF-beta and TNF-alpha regulation, and they further suggest a molecular mechanism for the opposing effects of these growth factors on gene expression.

The CD8 beta polypeptide is required for the recognition of an altered peptide ligand as an agonist.

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca2+ mobilization), and late (interleukin-2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 alpha and beta chains. With a video imaging system, we showed that the intracellular Ca2+ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca2+ concentration gradient which can be detected shortly after T cell interaction with antigen-presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8beta in the CD8 co-receptor dimers expressed at the T cell surface.

Peroxisome proliferator-activated receptor agonists.

The peroxisome proliferator-activated receptors are a family of three ligand-activated transcription factors. Fibrate antihyperlipidemic drugs and thiazolidinedione antihyperglycemic drugs were recently identified as synthetic ligands for these receptors. In addition, certain unsaturated fatty acids and eicosanoids were shown to bind the receptors, and thus represent naturally occurring PPAR ligands. The synthetic and natural ligands have proven to be powerful tools in dissecting the biology of these orphan receptors.

Tumor necrosis factor-alpha mediates changes in tissue protein turnover in a rat cancer cachexia model.

Rats bearing the Yoshida AH-130 ascites hepatoma showed enhanced fractional rates of protein degradation in gastrocnemius muscle, heart, and liver, while fractional synthesis rates were similar to those in non-tumor bearing rats. This hypercatabolic pattern was associated with marked perturbations of the hormonal homeostasis and presence of tumor necrosis factor in the circulation. The daily administration of a goat anti-murine TNF IgG to tumor-bearing rats decreased protein degradation rates in skeletal muscle, heart, and liver as compared with tumor-bearing rats receiving a nonimmune goat IgG. The anti-TNF treatment was also effective in attenuating early perturbations in insulin and corticosterone homeostasis. Although these results suggest that tumor necrosis factor plays a significant role in mediating the changes in protein turnover and hormone levels elicited by tumor growth, the inability of such treatment to prevent a reduction in body weight implies that other mediators or tumor-related events were also involved.