Molecular simplification of lipid A structure: TLR4-modulating cationic and anionic amphiphiles

A growing body of data suggests that therapies based on Toll-like receptors (TLR) targeting, in particular TLR4, holds promise in curing autoimmune and inflammatory pathologies still lacking specific treatment, included several rare diseases. While TLR4 activators (agonists) have already found successful clinical application as vaccine adjuvants, the use of TLR4 blockers (antagonists) as antisepsis agents or as agents against inflammatory diseases (including arthritis, multiple sclerosis, neuroinflammations) and cancer is still at a preclinical phase of development. This minireview focuses on recent achievements on the development of TLR4 modulators based on lipid A structure simplification, in particular on compounds having disaccharide or monosaccharide structures. As the TLR4 activity of natural TLR4 ligands (lipopolysaccharide, LPS and its biologically active part, the lipid A) depends on both the structure of endotoxin aggregates in solution and on single-molecule interaction with MD-2 and CD14 receptors, the rational design of TLR4 modulators should in principle take into account both these factors. In the light of the most recent advances in the field, in this minireview we discuss the structure–activity relationship in simplified lipid A analogs, with cationic or anionic amphiphilic structures.     

Physiologic responses to loud tones in Israeli patients with posttraumatic stress disorder.

Orbicularis oculi (eye blink) electromyogram, skin conductance, and heart rate responses to 15 consecutive 95-dB, 500-millisecond, 1000-Hz tones with 0-millisecond rise and fall times were measured in 14 patients with posttraumatic stress disorder, 14 patients with other anxiety disorders, 15 mentally healthy subjects with past traumatic experiences, and 19 mentally healthy subjects with no trauma history. The patients with posttraumatic stress disorder showed significantly larger skin conductance and heart rate responses and a trend toward larger electromyogram responses to the tones than every other group. These effects were not explained by subjective anxiety, resting physiologic arousal, physiologic arousal preceding the tone trials, or initial physiologic responsivity. The group with posttraumatic stress disorder was the only one that failed to show habituation of skin conductance responses.     

Metoclopramide effect on serum prolactin LH and FSH in patients with polycystic ovary syndrome

In order to investigate the DA activity in polycystic ovary syndrome (PCOS) we studied the response of LH, FSH and PRL to a dopamine receptor antagonist metoclopramide (MCP-10 mg iv) in 12 PCO subjects (7 with normal and 5 with elevated levels of prolactin). The prolactin and LH responses to metoclopramide were compared to those obtained in 6 normal cycling women. Although a significant increase in PRL levels was documented after MCP administration in all PCO patients and normal cycling women (p less than 0.01), the highest increment in PRL levels was observed in normoprolactinemic PCO subjects. In contrast a blunted PRL response was observed in hyperprolactinemic PCO patients. There was a negative correlation between basal PRL levels and the maximum net increase in PRL after MCP. In both groups of PCO subjects MCP administration caused initial decrease in LH levels followed by an increase after 4 h. In hyperprolactinemic PCO patients this observed MCP effect on LH was more pronounced and significantly different in comparison with normoprolactinemic PCO patients (p less than 0.01). MCP administration did not cause significant acute alterations in LH levels in normal cycling women and no significant FSH changes in either PCO or control subjects. A relative dopamine deficiency might cause hypersecretion of PRL and LH in patients with PCOS and hyperprolactinemia.     

ACTH-producing cells of 21-day-old rat fetuses after maternal dexamethasone exposure

Adrenocorticotrophic hormone (ACTH) is essential for developmental maturation of numerous organ systems during the fetal period and for adaptation to environmental challenges. Immunocytochemical and stereological methods were used in the present study to examine the effects of dexamethasone (Dx) administration during pregnancy on fetal rat pituitary ACTH-producing cells. Doses of 0.5, 0.5 and 1.0 mg Dx/kg body weight/day were given to the dams on 3 consecutive days starting on day 16 of gestation. Morphometric analysis of the ACTH-producing cells of fetuses at 21 days of gestation revealed significant inhibition by 24% and 27%, respectively, of cell volume and cell number after maternal Dx administration, whereas the volume of cell nuclei and volume density of ACTH-stained cells were insignificantly decreased. Immunocytochemical analysis showed reduced numbers, sizes and immunopositivity of ACTH cells of 21-day-old fetuses from Dx-treated dams as compared with the control group. Maternal Dx treatment in the period of intense differentiation of the hypothalamo-hypophyseal-adrenal system had an inhibitory effect on fetal function and proliferative activity of ACTH-producing cells at 21 days of gestation. Thus, inhibition of activity of fetal ACTH-producing cells may lead to adrenal suppression, modified activity of the hypothalamo-pituitary-adrenal axis and reduced body weight possibly causing lasting functional abnormalities.     

Effects of adrenalectomy and gonadectomy on sex and stress-induced differences in bicuculline-induced convulsions

The effects of adrenalectomy, gonadectomy and combined adrenalectomy plus gonadectomy on the previously described sex-dependent anticonvulsive effect of swim stress were studied in rats. The convulsive signs (myoclonic twitch, generalized convulsions, tonic hindlimb extension) were produced by constant i.v. infusion of γ-aminobutyric acid_A (GABA_A) antagonist bicuculline, which started 15 min after termination of swim stress (10-min swim at 18–19°C). Adrenalectomy decreased the threshold doses of bicuculline producing the first myoclonic twitch and the onset of generalized convulsions only in females. In adrenalectomized females, but not in males, swim stress enhanced the threshold dose of bicuculline producing generalized convulsions, but, unlike in adrenal-intact animals, it failed to enhance the dose of bicuculline producing tonic hindlimb extension. In gonadectomized stressed and unstressed animals all sex differences disappeared, and swim stress enhanced in both sexes only the threshold doses of bicuculline producing tonic hindlimb extension. Adrenalectomized plus gonadectomized animals displayed clear sex differences in doses of bicuculline necessary to produce all the convulsive signs. In the same animals swim stress postponed, especially in females, the onset of the first myoclonic twitch and generalized convulsions, but not the onset of tonic hindlimb extension. In summary, our results suggest that hormones of the adrenal and gonadal glands are only partly responsible for decreased susceptibility, especially of female rats, to the GABA_A antagonist bicuculline. Moreover, they have demonstrated that stress produces a gender-specific anticonvulsive effect even in the animals completely deprived of steroid hormones of peripheral origin. Received: 4 June 1998 / Accepted: 11 June 1999     

Estimation of genetic risk for type 1 diabetes

The most important gene loci defining risk of type 1 diabetes mellitus (T1DM) are located within the HLA gene region. HLA-DQ molecules are of primary importance but HLA-DR gene products modify the risk conferred by HLA-DQ. The risk associated with an HLA genotype is defined by the particular combination of susceptible and protective alleles. The highest risk is associated with a combination of two different risk haplotypes (7% risk to develop T1DM in Finland) whereas protective genotypes covering 69% of population have a risk of less than 0.2%). The complicated analysis of HLA genotypes is simplified by strong linkage disequilibrium between HLA-DRB1, -DQA1 and -DQB1 loci. In many cases one can deduce the alleles of other loci based on determination of the alleles in one locus. Differences between various populations in the frequency of marker alleles and in the linkages between them has to be taken into account. We have developed PCR based typing methods that utilize blood spot samples, microtiter plate format and lanthanide labeled oligonucleotide probes to define HLA-DQ and -DR alleles relevant for T1DM risk. Typing is run stepwise so that after initial HLA-DQB1 typing only those samples will be further analyzed in which -DQA1 or -DRB1 typing is informative and expected to contribute to the risk estimation. This method has been used to screen more than 50,000 newborn infants in Finland over a time period of 6 years, and it has been able to identify most children who have developed T1D during the follow-up period. The efficiency of the procedure has also been tested in Finnish and Greek populations.     

The long pentraxin PTX3 up-regulates tissue factor in activated monocytes: another link between inflammation and clotting activation

Pentraxin-3 (PTX3), an acute-phase protein that belongs to the family of the PTXs, is found elevated in septic shock and increased in patients with acute myocardial infarction. As tissue factor (TF) plays a key role in thrombosis and inflammation associated with atherosclerosis and as we have recently reported that PTX3 increases TF synthesis in endothelial cells, we tested whether PTX3 could modulate TF expression in monocytes. Monocytes from peripheral blood of healthy donors were incubated with highly purified PTX3 with or without lipopolysaccharide (LPS). Cells were then disrupted, and procoagulant activity was assessed by a one-stage clotting time. PTX3 enhanced TF activity and antigen from LPS-stimulated monocytes in a dose-dependent way. The effect was specific, as other PTXs, such as C-reactive protein and serum amyloid P component, were ineffective. Moreover, the increase in activity was specific for LPS, as in the presence of other TF-inducing agents such as interleukin-1beta and tumor necrosis factor alpha, PTX3 was not effective. The increase in TF activity requires mRNA synthesis, as assessed by polymerase chain reaction. The mechanism by which PTX3 modulates TF synthesis resides in an enhanced IkappaB, alpha phosphorylation and degradation and increased migration of the transacting factor c-Rel/p65 into the nucleus, as determined by Western blot and electro-mobility shift assay. These results show that PTX3 is an enhancer of the expression of TF by mononuclear cells. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. PTX3 increases TF expression, thus potentially playing a role in thrombogenesis and wound healing.     

Cytotoxicity on tumour cells of human mononuclear phagocytes: defective tumoricidal capacity of alveolar macrophages.

Human cells of the monocyte-macrophage lineage were isolated by adherence from peripheral blood, peritoneal exudate, non-neoplastic ascites, benign ovarian cystic fluid and bronchoalveolar lavages. Cytolytic activity was measured as 3H-thymidine release from prelabelled mKSA TU5 tumour cells over 48-72 hr and cytostasis was evaluated in a 72-hr spectrophotometric assay. Mononuclear phagocytes from the various anatomical sites examined, except lung alveolar spaces, were significantly cytolytic and cytostatic on target cells. Unlike other cells of the monocyte-macrophage lineage, alveolar macrophages were not cytocidal, but significantly inhibited tumour cell proliferative capacity. Peripheral blood monocytes and peritoneal macrophages showed enhanced cytotoxicity in the presence of partially purified human fibroblast interferon or of lymphokine supernatants from mitogen-stimulated lymphocytes. In contrast, interferon did not affect the cytotoxic potential of alveolar macrophages, whereas lymphokines augmented their cytostatic activity and rendered them weakly cytolytic.