Humoral immunity and correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis virus in children

Vaccination against tick-borne encephalitis (TBE) virus is the measure of choice for disease control in endemic areas, as no treatment is available. In Italy, the province of Belluno is one of the most active TBE virus infection foci. In this study sera were examined from vaccinated children living in areas around Belluno in order to monitor the immune response after anti-TBE vaccination. For the assessment of neutralizing antibodies, a plaque reduction neutralization test (PRNT) was optimized and the correlation between enzyme-linked immunosorbent assay (ELISA), hemaglutination inhibition (HI), and neutralizing antibodies titers was evaluated. All children had high serum levels of TBE IgG in ELISA test after the vaccination, in agreement with previous studies. HI and PRNT titers ranged between very low and high levels. A good correlation between HI and PRNT titers, and with IgG ELISA titers, was observed. PRNT is an useful assay for monitoring protective immunity after the completion of anti-TBE vaccination. This type-specific assay is an important tool for differential diagnosis in cases where the presence of cross-reactive antibodies due to other flavivirus infections or vaccinations cannot be excluded.     

Encapsulation, in vitro characterization, and in vivo biocompatibility of Sertoli cells in alginate-based microcapsules

A method for microencapsulation of isolated neonatal porcine Sertoli cells is described. Using a conventional alginate-poli-L-ornithine encapsulation procedure, which has been used in our laboratory for almost two decades to envelop pancreatic islets, we observed significant loss of Sertoli cell viability, possibly due to excessive Ca(2+) ion exposure. Replacing calcium with barium, or shortening the incubation period in the presence of Ca ions, we obtained barium or calcium alginate gel microbeads that did not alter morphology and viability of the encapsulated Sertoli cells. The procedure might permit access to a novel approach to immunologically alter cell graft acceptance.     

Soluble antigens from group B streptococci induce cytokine production in human blood cultures.

Group B streptococcal antigens stimulated tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 production in human blood cultures in a concentration- and time-dependent fashion. The minimal concentrations of type-specific polysaccharides, lipoteichoic acid, and group-specific polysaccharide required to produce these effects were, respectively, 0.01, 1, and 10 microg/ml. Cell separation experiments indicated that monocytes were the cell type mainly responsible for cytokine production. Time course studies indicated that TNF-alpha was released before the other cytokines. TNF-alpha, however, did not appear to directly induce IL-1beta, as shown by blockade experiments with anti-TNF-alpha antibodies. IL-6 levels were moderately but significantly decreased by anti-TNF-alpha. These data indicate that several products from group B streptococci are able to directly stimulate human monocytes to release TNF-alpha, IL-1beta, and IL-6. These findings may be clinically relevant, since proinflammatory cytokines can mediate pathophysiologic changes during sepsis.     

A double cryptic chromosome imbalance is an important factor to explain phenotypic variability in Wolf-Hirschhorn syndrome

A total of five Wolf-Hirschhorn syndrome (WHS) patient with a 4p16.3 de novo microdeletion was referred because of genotype-phenotype inconsistencies, first explained as phenotypic variability of the WHS. The actual deletion size was found to be about 12 Mb in three patients, 5 Mb in another one and 20 Mb in the last one, leading us to hypothesize the presence of an extrachromosome segment on the deleted 4p. A der(4)(4qter --> p16.1::8p23 --> pter) chromosome, resulting from an unbalanced de novo translocation was, in fact, detected in four patients and a der(4)(4qter --> q32::4p15.3 --> qter) in the last. Unbalanced t(4;8) translocations were maternal in origin, the rec(4p;4q) was paternal. With the purpose of verifying frequency and specificity of this phenomenon, we investigated yet another group of 20 WHS patients with de novo large deletions (n = 13) or microdeletions (n = 7) and with apparently straightforward genotype-phenotype correlations. The rearrangement was paternal in origin, and occurred as a single anomaly in 19 out of 20 patients. In the remaining patient, the deleted chromosome 4 was maternally derived and consisted of a der(4)(4qter --> 4p16.3::8p23 --> 8pter). In conclusions, we observed that 20% (5/25) of de novo WHS-associated rearrangements were maternal in origin and 80% (20/25) were paternal. All the maternally derived rearrangements were de novo unbalanced t(4;8) translocations and showed specific clinical phenotypes. Paternally derived rearrangements were usually isolated deletions. It can be inferred that a double, cryptic chromosome imbalance is an important factor for phenotypic variability in WHS. It acts either by masking the actual deletion size or by doubling a quantitative change of the genome.     

Inhibition of vascular endothelial growth factor (VEGF) signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts

Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. Vessels in Lewis lung tumors, which lacked endothelial fenestrations, showed less regression. In both tumors, pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment vessel number and location and a potential scaffold for vessel regrowth. The potent anti-vascular action observed is evidence that VEGF signaling inhibitors do more than stop angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel phenotype may be predictive of exceptional sensitivity to these inhibitors.     

Haemophilus influenzae porin induces Toll-like receptor 2-mediated cytokine production in human monocytes and mouse macrophages

The production of proinflammatory cytokines is likely to play a major pathophysiological role in meningitis and other infections caused by Haemophilus influenzae type b (Hib). Previous studies have shown that Hib porin contributes to signaling of the inflammatory cascade. We examined here the role of Toll-like receptors (TLRs) and the TLR-associated adaptor protein MyD88 in Hib porin-induced production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Hib porin-induced TNF-alpha and IL-6 production was virtually eliminated in macrophages from TLR2- or MyD88-deficient mice. In contrast, macrophages from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, which are defective in TLR4 function, responded normally to Hib porin. Moreover anti-TLR2 antibodies but not anti-TLR4 antibodies significantly reduced Hib porin-stimulated TNF-alpha and IL-6 release from the human monocytic cell line THP-1. These data indicate that the TLR2/MyD88 pathway plays an essential role in Hib porin-mediated cytokine production. These findings may be useful in the development of alternative therapies aimed at reducing excessive inflammatory responses during Hib infections.     

The role of nitrinergic connections in central cardiovascular responses mediated by physostigmine infused into posterior hypothalamus

In the last few decades, cholinergic connections located into posterior hypothalamus (PH) have been implicated in the central regulation of blood pressure (BP). Here we investigated the role of nitric oxide (NO) in the blood pressure response elicited by infusion of physostigmine into PH of normotensive rats. In freely moving rats, physostigmine (60-200 nM) produced a dose- and time-dependent elevation of BP which was antagonized by the antimuscarinic drug scopolamine (60 nM) and by L-NAME (100 microM), an inhibitor of NO synthase, both infused into the same site. In contrast, L-arginine (L-Arg; 100 microM), the precursor of NO, and glyceryltrinitrate (GTN; 140 nM), an NO donor, infused into the PH did not affect physostigmine-related pressor response. In rats pre-treated with Escherichia coli lipopolisaccharide (LPS; 0.5 microg i.p. 24h beforehand), however, scopolamine, L-Arg and GTN produced a decrease of BP, an effect antagonized by L-NAME. This suggests that NO only slightly modulates physostigmine-related pressor response elicited into PH of LPS-untreated rats. In contrast, the release of large amounts of NO generated by pre-treating rats with LPS, down-regulates cholinergic connections located at the PH, thus contributing in the central dysregulation of BP which can be found when high circulating endotoxin levels may occur.     

Could mitochondrial haplogroups play a role in sporadic amyotrophic lateral sclerosis?

Mitochondrial impairment has been implicated in the pathogenesis of the amyotrophic lateral sclerosis (ALS). Furthermore, mitochondrial-specific polymorphisms were previously related to other neurodegenerative diseases, such as Parkinson, Friedreich and Alzheimer disease. To investigate if specific genetic polymorphisms within the mitochondrial genome (mtDNA) could act as susceptibility factors and contribute to the clinical expression of sporadic ALS (sALS), we have genotyped predefined European mtDNA haplogroups in 222 Italian patients with sALS and 151 matched controls. Individuals classified as haplogroup I demonstrated a significant decrease in risk of ALS versus individuals carrying the most common haplogroup, H (odds ratio 0.08, 95% confidence interval 0.04-0.4, p < 0.01). Further stratification of the dataset by sex, age and site of onset of disease and survival failed to reach significance for association. Our study provides evidence of the contribution of mitochondrial variation to the risk of ALS development in Caucasians. Further it may help elucidate the mechanism of the mitochondrial dysfunction detectable in ALS, and may be of relevance in development of strategies for the treatment of this disease.