Secrets and lies of host-microbial interactions: MHC restriction and trans-regulation of T cell trafficking conceal the role of microbial agents on the edge between health and multifactorial/complex diseases.

Here we critically discuss data supporting the view that microbial agents (pathogens, pathobionts or commensals alike) play a relevant role in the pathogenesis of multifactorial diseases, but their role is concealed by the rules presiding over T cell antigen recognition and trafficking. These rules make it difficult to associate univocally infectious agents to diseases' pathogenesis using the paradigm developed for canonical infectious diseases. (Cross-)recognition of a variable repertoire of epitopes leads to the possibility that distinct infectious agents can determine the same disease(s). There can be the need for sequential infection/colonization by two or more microorganisms to develop a given disease. Altered spreading of infectious agents can determine an unwanted activation of T cells towards a pro-inflammatory and trafficking phenotype, due to differences in the local microenvironment. Finally, trans-regulation of T cell trafficking allows infectious agents unrelated to the specificity of T cell to modify their homing to target organs, thereby driving flares of disease. The relevant role of microbial agents in largely prevalent diseases provides a conceptual basis for the evaluation of more specific therapeutic approaches, targeted to prevent (vaccine) or cure (antibiotics and/or Biologic Response Modifiers) multifactorial diseases.

Application of capsular sequence typing (CST) to serotype non-viable Streptococcus pneumoniae isolates from an old collection.

Serotyping of Streptococcus pneumoniae is essential for monitoring changes in the pneumococcal population and the impact of vaccines. Recently, various DNA-based methods have become available and are increasingly used because they are cheaper and easier to perform than the Quellung reaction. Our aim was to apply a DNA-based method, capsular sequence typing (CST), to a collection of non-viable lyophilized pneumococcal isolates dating from the 1980s to elucidate the serotypes circulating in Italy 30 years ago. As a preliminary evaluation of the method, CST was applied to 68 recent pneumococcal isolates representative of the most common serotypes circulating in Italy in invasive pneumococcal disease (IPD) previously serotyped by the Quellung reaction. CST was then applied to 132 lyophilized non-viable isolates. A serotype-specific polymerase chain reaction (PCR), using primers suggested by the Centers for Disease Control and Prevention (CDC), was performed when CST did not yield a univocal serotype. Considering the control isolates, CST concordance with the Quellung reaction was 95.6 %. For the non-viable lyophilized isolates, CST identified a univocal serotype for 59.4 % of the isolates. This percentage increased to 78.1 % if CST was combined with serotype-specific PCR. The most frequent serotypes in the collection of non-viable strains were: 3 (15.6 %), 14 (11.7 %), 35B (5.5 %), 19A (5.5 %), and 8 (4.7 %). CST proved to be a valid method for serotyping pneumococcal strains and provided information about pneumococcal serotypes present in Italy 30 years ago. The combination of CST with serotype-specific PCR was an effective strategy to identify pneumococcal serotypes that can be suggested also for routine laboratories.

CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease.

OBJECTIVES: To analyze the diagnostic sensitivity and specificity of various brain-derived proteins (14-3-3, Tau, neuron specific enolase [NSE], and S100b) in the CSF of patients with Creutzfeldt-Jakob disease (CJD) and to analyze biologic factors that modify these parameters. METHODS: CSF was tested for 14-3-3, Tau, NSE, and S100b in 1,859 patients with sporadic, genetic, iatrogenic, and variant CJD, and in 1,117 controls. RESULTS: The highest sensitivity was achieved for 14-3-3 and Tau in sporadic CJD (85% and 86%), and a combined determination of 14-3-3 and Tau, S100b, or NSE increased the sensitivity to over 93%. A multivariate analysis showed that the sensitivity of all tests was highest in patients with the shortest disease duration, age at onset >40 years, and homozygosity at codon 129 of the prion protein gene. In a group of patients with repeated lumbar punctures, a second test also increased the diagnostic sensitivity. CONCLUSIONS: The detection of elevated levels of brain-derived proteins in the CSF in patients with suspected Creutzfeldt-Jakob disease is a valuable diagnostic test. A second lumbar puncture may be of value in patients with atypical clinical course in whom the first test was negative.

Calcium channels are present in the apical plasma membranes of the hepatopancreatic B-cells of Marsupenaeus japonicus.

This study demonstrates the existence of calcium channels in the apical membranes of the hepatopancreatic blister (B) cells of Marsupenaeus japonicus. Using brush-border membrane vesicles we demonstrated that the channel-mediated calcium passive flux was saturable and was stimulated by a transmembrane electrical potential difference and inhibited by barium. We raised a monoclonal antibody (Mab 24A4) against the calcium channel, which allowed us to inhibit the channel-mediated calcium uptake. By immunocytochemistry, using Mab 24A4, we demonstrated that these channels are located at the apical membrane of hepatopancreatic B cells. Finally, by measuring the calcium uptake in R- and B-enriched cell suspensions, we showed that only the plasma membrane of the B cells expresses a channel-mediated calcium uptake inhibited by barium, verapamil and the monoclonal antibody 24A4. The plasma membrane of R cells did not show calcium channels.

Differential expression of Na+/D-glucose cotransport in isolated cells of Marsupenaeus japonicus hepatopancreas.

D-Glucose absorptive processes at the gastrointestinal tract of decapod crustaceans are largely under-investigated. We have studied Na(+)-dependent D-glucose transport (Na(+)/D-glucose cotransport) in the hepatopancreas of the Kuruma prawn, Marsupenaeus japonicus, using both brush-border membrane vesicles and purified R and B hepatopancreatic cell suspensions. As assessed by brush-border membrane vesicle studies, Na(+)/D-glucose cotransport was inhibited by phloridzin and responsive to the (inside negative) membrane potential. Furthermore, it was strongly activated by protons (although only in the presence of an inside-negative membrane potential), which correlates with the fact that the lumen of crustacean hepatopancreatic tubules is acidic. When assayed in purified R and B cell suspensions, Na(+)/D-glucose cotransport activity was restricted to B cells only. Mab 13, a monoclonal antibody recognizing an 80- to 85-KDa protein at the brush-border membrane location, inhibited Na(+)/D-glucose cotransport in brush-border membrane vesicles as well as in enriched B cell suspensions. Primers designed after comparison of highly homologous regions of various mammalian sodium-glucose transporter) nucleotide sequences failed to produce RT-PCR amplification products from Kuruma prawn hepatopancreatic RNA. The molecular nature of this Na(+)/D-glucose cotransport system is still to be established.

D-glucose transport in decapod crustacean hepatopancreas.

Physiological mechanisms of gastrointestinal absorption of organic solutes among crustaceans remain severely underinvestigated, in spite of the considerable relevance of characterizing the routes of nutrient absorption for both nutritional purposes and formulation of balanced diets in aquaculture. Several lines of evidence attribute a primary absorptive role to the digestive gland (hepatopancreas) and a secondary role to the midgut (intestine). Among absorbed organic solutes, the importance of D-glucose in crustacean metabolism is paramount. Its plasma levels are finely tuned by hormones (crustacean hyperglycemic hormone, insulin-like peptides and insulin-like growth factors) and the function of certain organs (i.e. brain and muscle) largely depends on a balanced D-glucose supply. In the last few decades, D-glucose absorptive processes of the gastrointestinal tract of crustaceans have been described and transport mechanisms investigated, but not fully disclosed. We briefly review our present knowledge of D-glucose transport processes in the crustacean hepatopancreas. A discussion of previous results from experiments with hepatopancreatic epithelial brush-border membrane vesicles is presented. In addition, recent advances in our understandings of hepatopancreatic D-glucose transport are shown, as obtained (1) after isolation of purified R-, F-, B- and E-cell suspensions from the whole organ by centrifugal elutriation, and (2) by protein expression in hepatopancreatic mRNA-injected Xenopus laevis oocytes. In a perspective, the applicability of these novel methods to the study of hepatopancreatic absorptive function will certainly improve our knowledge of this structurally complex organ.

An L-proline-dependent proton flux is located at the apical membrane level of the eel enterocytes.

This study has demonstrated the existence of an L-proline-dependent (Na independent) proton flux at the apical membrane level of the eel intestinal absorbing cells. Using isolated eel enterocytes and the pH-sensitive fluorescent dye 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF), it was shown that a 20 mM concentration of the imino acid L-proline in the extracellular medium determined an intracellular acidification of approximately 0.28 pH units. However, neither sucrose nor other amino acids were able to significantly acidify the resting intracellular pH. A hyperbolic relationship between extracellular proline concentration and intracellular proton accumulation was observed. Using both isolated brush-border and basolateral membrane vesicles, it was demonstrated that this proline-proton cotransport mechanism was located at the apical membrane level only. In addition, the existence of a coupling mechanism between proline and proton fluxes was demonstrated by the observation that, in brush-border membrane vesicles, the presence of a pH gradient (pH(in) > pH(out)) stimulated the uptake of L-proline.

Electroneutral Na+/H+ exchange in brush-border membrane vesicles from Penaeus japonicus hepatopancreas.

An electroneutral Na+/H+ exchange mechanism (dimethylamiloride inhibitable, Li+ sensitive, and Ca2+ insensitive) was identified in brush-border membrane vesicles (BBMV) from Kuruma prawn hepatopancreas by monitoring Na(+)-dependent H+ fluxes with the pH-sensitive dye acridine orange and measuring 22Na+ uptake. Kinetic parameters measured under short-circuited conditions were the Na+ concentration that yielded one-half of the maximal dissipation rate (Fmax) of the preset transmembrane delta pH (KNa) = 15 +/- 2 mM and Fmax = 3,626 +/- 197 delta F.min-1.mg protein-1, with a Hill coefficient for Na+ of approximately 1. In addition, the inhibitory constant for dimethylamiloride was found to be approximately 1 microM. The electroneutral nature of the antiporter was assessed in that an inside-negative transmembrane electrical potential neither affected kinetic parameters nor stimulated pH-dependent (intracellular pH > extracellular pH) 22Na+ uptake. In contrast, electrogenic pH-dependent 22Na+ uptake was observed in lobster hepatopancreatic BBMV. Substitution of chloride with gluconate resulted in increasing KNa and decreasing delta Fmax, which suggests a possible role of chloride in the operational mechanism of the antiporter. These results indicate that a Na+/H+ exchanger, resembling the electroneutral Na+/H+ antiporter model, is present in hepatopancreatic BBMV from the Kuruma prawn Penaeus japonicus.

An electroneutral anion exchange mechanism is present in brush-border membranes isolated from eel kidney.

The mechanism of bicarbonate translocation across the luminal membrane of the eel (Anguilla anguilla) kidney tubular cells was studied by monitoring the uptake of H14CO3- into isolated brush-border membrane vesicles. Results indicate that the presence of a transmembrane outwardly directed Cl- gradient was able to transiently accumulate H14CO3- into the vesicular space, whereas neither an inwardly directed sodium gradient nor a transmembrane electrical potential gradient (inside positive) was able to stimulate the H14CO3- influx. This anion-dependent H14CO3- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that an anion exchanger was present in the brush-border membrane vesicles.

A monoclonal antibody to mammalian angiotensin II AT1 receptor recognizes one of the angiotensin II receptor isoforms expressed by the eel (Anguilla anguilla).

Using labelled ligand-binding methods, previous studies have identified specific angiotensin II receptors (Ang II-Rs) in eel liver, kidney and intestine membranes. Isoelectric focusing on polyacrylamide gels also showed that there are two Ang II-R isoforms in eel liver, focusing at isoelectric points (pI) 6.5 and 6.7. These may have different functions. In contrast, eel enterocyte plasma membrane and renal brush border membranes contain only the pI 6.5 form. To characterize the eel receptors more fully, a newly developed monoclonal antibody (6313/G2) which selectively recognizes the AT1 subtype of mammalian Ang II-R was used. In ligand-binding experiments, the preincubation of eel liver membranes with 6313/G2 antibody eliminated the specific [3,5-3H]Tyr4-Ile5-Ang II binding. Moreover, Ang II-receptor complexes from solubilized liver membranes, which were immunoprecipitated by 6313/G2-coated beads, had a pI of 6.5. In immunoblotting experiments, the antibody recognized the isoform focusing at pI 6.5 in eel intestine and liver preparations, but not the liver pI 6.7 isoform. Immunoblotting of SDS gels showed that the antibody bound to a single protein of molecular mass of 75 kDa in eel liver, gill and kidney and to a doublet of molecular mass of about 74 and 75 kDa in intestinal membrane preparations. Immunocytochemistry of paraffin-embedded and cryostat sections of eel liver, kidney, intestine and gill showed that antibody 6313/G2 bound to uniformly distributed intracellular sites and cell surface membranes in proximal tubular cells, absorptive intestinal cells, hepatocytes and chloride cells. It also stained endothelium and both the longitudinal and circular layers of smooth muscle cells in the intestine. The data suggest that the previously described Ang II-R from eel liver, kidney and intestine may be similar to the mammalian AT1 subtype.

Angiotensin II stimulation of the basolateral located Na+/H+ antiporter in eel (Anguilla anguilla) enterocytes.

The pH-sensitive fluorescent dye, 2',7'-bis-carboxyethyl-5, 6-carboxyfluorescein acetoxymethyl ester, was used to examine the effects of fish or human angiotensin II (Ang II) on the activity of the basolateral located Na+/H+ antiporter in eel intestinal cell suspensions. Exposure of eel enterocytes to either hormone led to an increased activity of the antiporter. This time- and dose-dependent stimulatory effect was inhibited by the specific antiporter inhibitor dimethylamiloride (DMA). Preincubation with a monoclonal antibody (6313/ G2), directed against the N-terminal extracellular domain of the mammalian AT1 Ang II receptor, prevented the stimulatory effect of the hormone and inhibited the binding of [3,5-3H] Tyr4-Ile5-Ang II to intestinal cell suspensions, suggesting specific binding of the antibody to the eel Ang II receptor. The results indicate that both fish and human Ang II stimulate the DMA-sensitive Na+/H+ antiporter present in eel intestinal cells by means of a mammalian AT1-like receptor.

Congo red prolongs the incubation period in scrapie-infected hamsters.

In scrapie-infected cells, Congo red inhibits both the replication of the infectious agent and accumulation of the protease-resistant form of PrP (PrP-res). In this report, we show that Congo red prolongs the incubation periods of hamsters experimentally infected with two different strains of scrapie.

Amphotericin B treatment dissociates in vivo replication of the scrapie agent from PrP accumulation.

Scrapie and related animal and human disorders are neurodegenerative diseases characterized by the formation of a modified, partly proteinase-resistant protein (PrP) of the host, which tends to aggregate as amyloid fibrils and accumulate in the brain of infected individuals. There is a general consensus that the pathological form of PrP (PrPSc) is essential for the clinical appearance of the disease, but whether it is part of the scrapie agent or a by-product of viral infection is still controversial. Here we report that treatment of scrapie-infected hamsters with amphotericin B delays the accumulation in the brain of the proteinase-resistant portion of PrPSc by about 30 days without affecting scrapie replication. The consequence is that hamsters treated with amphotericin B developed clinical signs of disease later than infected controls. We argue that the proteinase-resistant portion of PrPSc is necessary for the development of the disease but that it is unlikely to be essential for scrapie replication.

Sulphate polyanions prolong the incubation period of scrapie-infected hamsters.

The effect of the organic sulphated polyanions, pentosan sulphate (SP54), dextran sulphate 500 (DS500) and suramin, have been tested on golden Syrian hamsters infected with the 263K strain of scrapie by the intraperitoneal (i.p.) or the intracerebral route. SP54 had the greatest effect in prolonging the incubation period of the disease when administered within 2 h of the i.p. inoculum. The same amount of SP54 given 24 h after scrapie inoculation had a potent effect in some animals and no effect in others. This result suggests that SP54 inhibits the uptake of the scrapie agent into the nerve endings and/or carrier cells at the site of the inoculum, i.e. the peritoneum, and that this event occurs in about 24 h. DS500 had a similar although less potent effect (22.4 days delay during the incubation period) than SP54 (54.4 days) when administered within 2 h of scrapie injection by the i.p. route, and suramin had only a minimal effect (10 days). This study suggests that treatment of scrapie and related spongiform encephalopathies of animals and man is possible only before the agent has reached the clinical target areas of the brain.

Experimental drug treatment of scrapie: a pathogenetic basis for rationale therapeutics.

Pharmacological treatment with polyanions or amphotericin B in hamsters with experimental scrapie reveals that it is possible to delay the appearance of the disease only when the drug is given before the invasion of the agent into the clinical target areas of the brain. We suggest such early treatment may be possible for individuals at high risk of acquiring the disease, such as healthy mutation-positive relatives of patients with familial Creutzfeldt-Jakob disease or Gerstmann-Sträussler syndrome, or recipients of potentially contaminated pituitary-extracted human growth hormone.

Measurement of the concentration of amphotericin B in brain tissue of scrapie-infected hamsters with a simple and sensitive method.

A simple, sensitive, and reproducible assay for the measurement of the amphotericin B concentration in tissue extracts was developed by using the fourth derivative of the absorption spectrum of amphotericin B between wavelengths of 330 and 430 nm. The amphotericin B concentration in spleen and brain was proportional to the total amount administered. The amphotericin B concentration in the brain was highly correlated with the increase in the mean incubation period of intracerebrally scrapie-infected hamsters.