Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K_d: 5 × 10⁻⁸ M) than [Tyr³⁶]PTHrP(1–36)amide or [Arg^11,13,Tyr³⁶]PTHrP(1–36)amide (apparent K_d for both: 2 × 10⁻⁹ M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶,Cys³⁸]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K_ds: 5 × 10⁻⁸ M and 8 × 10⁻⁸ M), while that of [Nle^8,18,Tyr³⁴]bPTH(7–34)amide was more than 10-fold lower (apparent K_d: 2 × 10⁻⁶ M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly¹².     

Characterization of thermostable α-glucosidase from Clostridium thermohydrosulfuricum 39E

Summary Clostridium thermohydrosulfuricum 39E produced a cell-bound -glucosidase. It was partially purified 140-fold by solubilizing with Triton X-100, ammonium sulfate treatment, DEAE-Sepharose CL-6B, octyl-Sepharose and acarbose-Sepharose affinity chromatography. The optimum temperature for the action of the enzyme was at 75°C. It had a half-life of 35 min at 75°C, 110 min at 70°C and 46 h at 60°C. The enzyme was stable at pH 5.0–6.0 and had an optimum pH at 5.0–5.5. It hydrolyzed the -1,4-linkages in maltose, maltotriose, maltotetraose and maltohexaose, the rate decreasing in order of higher-sized oligosaccharides. The enzyme preparation also hydrolyzed the -1,6 linkages in isomaltose and isomaltotriose. It rapidly hydrolyzed p-nitrophenyl -d-glucoside (pNPG). The K _m values for maltose, isomaltose, panose, maltotriose, and pNPG were 1.85, 2.95, 1.72, 0.58, and 0.31 mm, respectively, at pH 5.5 and 60°C. The enzyme produced glucose from all these substrates. The enzyme preparation did not require any metal ion for activity. The -glucosidase activity was inhibited by acarbose. Offprint requests to: B. C. Saha     

Development of highly organized lymphoid structures in Buruli ulcer lesions after treatment with rifampicin and streptomycin

Background

Buruli ulcer caused by Mycobacterium ulcerans is an infection of the subcutaneous tissue leading to chronic necrotising skin ulcers. The pathogenesis is associated with the cytocidal and immunosuppressive activities of a macrolide toxin. Histopathological hallmark of progressing disease is a poor inflammatory response despite of clusters of extracellular bacilli. While traditionally wide excision of the infected tissue was the standard treatment, provisional WHO guidelines now recommend an eight week pre-treatment with streptomycin and rifampicin.

Methodology/Principal Findings

We conducted a detailed immunohistochemical analysis of tissue samples from Buruli patients who received antibiotic treatment. Cellular immune response along with bacterial load and distribution were monitored. We demonstrate that this treatment leads to the development of highly organized cellular infiltration surrounding areas of coagulative necrosis. Diffuse infiltrates, granulomas and dense lymphocyte aggregation close to vessels were observed. Mycobacterial material was primarily located inside mononuclear phagocytes and microcolonies consisting of extracellular rod-shaped mycobacteria were no longer found. In observational studies some patients showed no clinical response to antibiotic treatment. Corresponding to that, one of five lesions analysed presented with huge clusters of rod-shaped bacilli but no signs of infiltration.

Conclusions/Significance

Results signify that eight weeks of antibiotic treatment reverses local immunosuppression and leads to an active inflammatory process in different compartments of the skin. Structured leukocyte infiltrates with unique signatures indicative for healing processes developed at the margins of the lesions. It remains to be analysed whether antibiotic resistance of certain strains of M. ulcerans, lacking patient compliance or poor drug quality are responsible for the absent clinical responses in some patients. In future, analysis of local immune responses could serve as a suitable surrogate marker for the efficacy of alternative treatment strategies.     

Production of mannitol by Lactobacillus intermedius NRRL B-3693 in fed-batch and continuous cell-recycle fermentations

Improved fermentation processes were developed for the production of mannitol by a heterofermentative lactic acid bacterium (Lactobacillus intermedius NRRL B-3693). A fed-batch fermentation protocol overcame limitations caused by high substrate concentrations. The process was developed using corn steep liquor and glucose as inexpensive industrial nutrient sources, supplemented with a small amount of soy peptone and manganese. The fed-batch process resulted in a concentration of 176 ± 0.5 g mannitol from 184 ± 0 g fructose and 92 ± 0.1 g glucose per L of final fermentation broth in 30 h with a volumetric productivity of 5.9 g/(L h). Further increases in volumetric productivity of mannitol were obtained in a continuous cell-recycle fermentation process that reached more than 40 g/(L h), despite reduced mannitol levels of 78–98 g/L and residual substrate of 10–20 g/L. This is the first report of such a high volumetric productivity of mannitol by a heterofermentative lactic acid bacterium.     

Skeletons in the p53 tumor suppressor closet: genetic evidence that p53 blocks bone differentiation and development

A series of in vitro tissue culture studies indicated that the p53 tumor suppressor promotes cellular differentiation, which could explain its role in preventing cancer. Quite surprisingly, however, two new in vivo studies provide genetic evidence that p53 blocks osteoblast differentiation and bone development. These interesting results and their biological and clinical implications are the focus of this comment.     

Genetic diversity in Mycobacterium ulcerans isolates from Ghana revealed by a newly identified locus containing a variable number of tandem repeats

The molecular typing methods used so far for Mycobacterium ulcerans isolates have not been able to identify genetic differences among isolates from Africa. This apparent lack of genetic diversity among M. ulcerans isolates is indicative of a clonal population structure. We analyzed the genetic diversity of 72 African isolates, including 57 strains from Ghana, by variable number of tandem repeat (VNTR) typing based on a newly identified polymorphic locus designated ST1 and the previously described locus MIRU 1. Three different genotypes were found in Ghana, demonstrating for the first time the genetic diversity of M. ulcerans in an African country. While the ST1/MIRU 1 allele combination BD/BAA seems to dominate in Africa, it was only rarely found in isolates from Ghana, where the combination BD/B was dominant and observed in all districts studied. A third variant genotype (C/BAA) was found only in the Amansie-West district. The results indicate that new genetic variants of M. ulcerans emerged and spread within Ghana and support the potential of VNTR-based typing for genotyping of M. ulcerans.     

Production and release of heat-labile toxin by wild-type human-derived enterotoxigenic Escherichia coli

Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals.     

Human colostrum contains IgA antibodies reactive to colonization factors I and II of enterotoxigenic Escherichia coli

Diarrhea is an important cause of morbidity and mortality amongst infants of low socio-economic levels in developing countries and in travelers who visit such areas. Enterotoxigenic E. coli strains express two sets of virulence-associated factors: enterotoxins (heat-stable toxins or heat-labile toxins) and colonization factors. Studies have shown that breast-feeding protects infants against infectious diseases, such as diarrhea, as it presents a great variety of immunological components. The aim of this study was to analyze the reactivity of immunoglobulin A from human colostrum to colonization factor antigens I and II. The colostrum ability in preventing enterotoxigenic E. coli adhesion to Caco-2 cells was also evaluated. Colostrum samples were collected from 32 healthy women, and a human colostrum pool was prepared. Enterotoxigenic E. coli strains expressing colonization factor antigens I and II were utilized. The colostrum pool and individual samples showed variable antienterotoxigenic E. coli immunoglobulin A titers, that were reactive with colonization factor antigen I and CS1/CS3 (colonization factor antigen II). The human colostrum pool and individual samples inhibited enterotoxigenic E. coli colonization factor antigen I and II adhesion to Caco-2 cells, at variable levels, and this ability was a result of immunoglobulin A antibodies reactive to these colonization factors. The immunoglobulin A-depleted pool lost this inhibitory ability. As bacterial adhesion is the initial mechanism of enterotoxigenic E. coli infection, breast-feeding could protect the offspring against diarrhea caused by this agent.     

On immunotherapies and cancer vaccination protocols: A mathematical modelling approach

In this paper we develop a new mathematical model of immunotherapy and cancer vaccination, focusing on the role of antigen presentation and co-stimulatory signaling pathways in cancer immunology. We investigate the effect of different cancer vaccination protocols on the well-documented phenomena of cancer dormancy and recurrence, and we provide a possible explanation of why adoptive (i.e. passive) immunotherapy protocols can sometimes actually promote tumour growth instead of inhibiting it (a phenomenon called immunostimulation), as opposed to active vaccination protocols based on tumour-antigen pulsed dendritic cells. Significantly, the results of our computational simulations suggest that elevated numbers of professional antigen presenting cells correlate well with prolonged time periods of cancer dormancy.