A Murine,Bispecific Monoclonal Antibody Simultaneously Recognizing β-Glucan and MP65 Determinants in Candida Species

There is a real medical need of new diagnostic tools for the early recognition of invasive Candida infections. We exploited a rather simple and rapid redox methodology to construct a bispecific monoclonal antibody (bsmAb) that combines a monoclonal antibody (mAb) directed against 1,3-β-D-glucan, a well-known, pan-fungal diagnostic biomarker, with a mAb recognizing MP65, a major immunogenic mannoprotein secreted by C.albicans and other Candida species. The bsmAb (MP65/bglu mAb) was successfully produced and purified at high yields and proved to bind and reveal simultaneously, with high sensitivity, the β-glucan and MP65 antigens in both purified and native forms. The MP65/bglu mAb is the first bispecific antibody generated against a fungal microorganism and may prove useful for the concurrent detection of different and clinically significant Candida biomarkers in patient sera.     

Antifungal activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fermentation: identification of novel compounds and long-term effect during storage of wheat bread

This study aimed at investigating the antifungal activity of Wickerhamomyces anomalus and sourdough lactic acid bacteria to extend the shelf life of wheat flour bread. The antifungal activity was assayed by agar diffusion, growth rate inhibition, and conidial germination assays, using Penicillium roqueforti DPPMAF1 as the indicator fungus. Sourdough fermented by Lactobacillus plantarum 1A7 (S1A7) and dough fermented by W. anomalus LCF1695 (D1695) were selected and characterized. The water/salt-soluble extract of S1A7 was partially purified, and several novel antifungal peptides, encrypted into sequences of Oryza sativa proteins, were identified. The water/salt-soluble extract of D1695 contained ethanol and, especially, ethyl acetate as inhibitory compounds. As shown by growth inhibition assays, both water/salt-soluble extracts had a large inhibitory spectrum, with some differences, toward the most common fungi isolated from bakeries. Bread making at a pilot plant was carried out with S1A7, D1695, or a sourdough started with a combination of both strains (S1A7-1695). Slices of the bread manufactured with S1A7-1695 did not show contamination by fungi until 28 days of storage in polyethylene bags at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate. The effect of sourdough fermentation with W. anomalus LCF1695 was also assessed based on rheology and sensory properties.     

Plant production of anti-β-glucan antibodies for immunotherapy of fungal infections in humans

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.     

Circadian rhythms of gastrointestinal function are regulated by both central and peripheral oscillators

Circadian clocks are responsible for daily rhythms in a wide array of processes, including gastrointestinal (GI) function. These are vital for normal digestive rhythms and overall health. Previous studies demonstrated circadian clocks within the cells of GI tissue. The present study examines the roles played by the suprachiasmatic nuclei (SCN), master circadian pacemaker for overt circadian rhythms, and the sympathetic nervous system in regulation of circadian GI rhythms in the mouse Mus musculus. Surgical ablation of the SCN abolishes circadian locomotor, feeding, and stool output rhythms when animals are presented with food ad libitum, while restricted feeding reestablishes these rhythms temporarily. In intact mice, chemical sympathectomy with 6-hydroxydopamine has no effect on feeding and locomotor rhythmicity in light-dark cycles or constant darkness but attenuates stool weight and stool number rhythms. Again, however, restricted feeding reestablishes rhythms in locomotor activity, feeding, and stool output rhythms. Ex vivo, intestinal tissue from PER2::LUC transgenic mice expresses circadian rhythms of luciferase bioluminescence. Chemical sympathectomy has little effect on these rhythms, but timed administration of the β-adrenergic agonist isoproterenol causes a phase-dependent shift in PERIOD2 expression rhythms. Collectively, the data suggest that the SCN are required to maintain feeding, locomotor, and stool output rhythms during ad libitum conditions, acting at least in part through daily activation of sympathetic activity. Even so, this input is not necessary for entrainment to timed feeding, which may be the province of oscillators within the intestines themselves or other components of the GI system.     

Circadian regulation of bird song, call, and locomotor behavior by pineal melatonin in the zebra finch

As both a photoreceptor and pacemaker in the avian circadian clock system, the pineal gland is crucial for maintaining and synchronizing overt circadian rhythms in processes such as locomotor activity and body temperature through its circadian secretion of the pineal hormone melatonin. In addition to receptor presence in circadian and visual system structures, high-affinity melatonin binding and receptor mRNA are present in the song control system of male oscine passeriform birds. The present study explores the role of pineal melatonin in circadian organization of singing and calling behavior in comparison to locomotor activity under different lighting conditions. Similar to locomotor activity, both singing and calling behavior were regulated on a circadian basis by the central clock system through pineal melatonin, since these behaviors free-ran with a circadian period and since pinealectomy abolished them in constant environmental conditions. Further, rhythmic melatonin administration restored their rhythmicity. However, the rates by which these behaviors became arrhythmic and the rates of their entrainment to rhythmic melatonin administration differed among locomotor activity, singing and calling under constant dim light and constant bright light. Overall, the study demonstrates a role for pineal melatonin in regulating circadian oscillations of avian vocalizations in addition to locomotor activity. It is suggested that these behaviors might be controlled by separable circadian clockworks and that pineal melatonin entrains them all through a circadian clock.