Tissue therapy with autologous dermal and epidermal culture cells for diabetic foot ulcers

A great part of diabetic ulcers on the lower extremities have difficult healing and represent the most common cause of non-traumatic amputation In case of patients unresponsive to the classical therapy with debridement, dressings and systemic antibiotic therapy, cell therapy may be an excellent indication. The objective of this study was to assess the efficacy of autologous skin cell (fibroblasts and keratinocytes) implants cultivated ex vivo and applied to long-standing (9?C34?years) skin ulcers of five diabetic patients (4 DM2 and 1 DM1) with autologous fibrin glue. There were six ulcers of onset between 4?months and 20?years before and from 4.0 to 36.62?cm² in size, located on the lower limbs and unresponsive to the several conventional treatments. Complete healing was observed in five ulcers (83.3%), after 21?C120?days. The patient who presented the largest ulcer had partial improvement in 40?days. It is believed that the more distal ulcer location is, the worse is its prognosis. There probably is a correlation between healing time, ulcer size and prior duration. No adverse reactions derived from the treatment occurred. It is concluded that this method is an excellent therapeutic option for diabetic ulcers, allowing faster healing. Its great advantage is being a minimally invasive procedure that can be carried out in an outpatient clinic.     

Airborne pollen patterns in Mar del Plata atmosphere (Argentina) and its relationship with meteorological conditions

This work is part of a series of aerobiological researches conducted in the city of Mar del Plata, Argentina. The annual, seasonal and daily features of the pollen cloud were analyzed over 2 years, together with the effects of the meteorological variables. Cupressaceae amounted to 75 and 54% of the annual total in each year, and it was analyzed separately due to its proven overrepresentation. Eighty-five pollen types were registered during the 2 years, bearing a similar annual pattern (more than 50% from August to November, December also being an important month). Arboreal pollen predominated between late winter and spring (61 and 49% of the annual total), while non-arboreal pollen did so from late spring to summer. The observed seasonality was significantly correlated with the monthly mean temperatures and associated with its effect on phenology. No significant differences along the day were detected between years and 50% of the daily total was recorded between 1000 and 1600 hours. The maximum concentration was registered at 1000 hours in spring and summer, but later in autumn and winter. Regarding the hourly scale, the significant correlation of total pollen with temperature and wind speed was positive, albeit negative with relative humidity. These effects are linked to airborne pollen release and transport. The role rainfall plays on airborne pollen is discussed. To interpret the results, it is important to consider the time scale at which the aerobiological phenomenon is analyzed.     

Increased Serum Hepcidin Levels in Subjects with the Metabolic Syndrome: A Population Study

The recent discovery of hepcidin, the key iron regulatory hormone, has changed our view of iron metabolism, which in turn is long known to be linked with insulin resistant states, including type 2 diabetes mellitus and the Metabolic Syndrome (MetS). Serum ferritin levels are often elevated in MetS (Dysmetabolic hyperferritinemia - DHF), and are sometimes associated with a true mild-to-moderate hepatic iron overload (dysmetabolic iron overload syndrome - DIOS). However, the pathophysiological link between iron and MetS remains unclear. This study was aimed to investigate, for the first time, the relationship between MetS and hepcidin at population level. We measured serum hepcidin levels by Mass Spectrometry in 1,391 subjects from the Val Borbera population, and evaluated their relationship with classical MetS features. Hepcidin levels increased significantly and linearly with increasing number of MetS features, paralleling the trend of serum ferritin. In multivariate models adjusted for relevant variables including age, C-Reactive Protein, and the HFE C282Y mutation, ferritin was the only significant independent predictor of hepcidin in males, while in females MetS was also independently associated with hepcidin. Overall, these data indicate that the fundamental iron regulatory feedback is preserved in MetS, i.e. that hepcidin tends to progressively increase in response to the increase of iron stores. Due to recently discovered pleiotropic effects of hepcidin, this may worsen insulin resistance and contribute to the cardiovascular complications of MetS.     

Mannosylated LigANI Produced in Pichia pastoris Protects Hamsters Against Leptospirosis

The C-terminal region of the Leptospiral immunoglobulin-like A protein (LigA) contains six carboxy-terminal Ig-like repeat domains (LigANI). Subunit vaccine preparations based on recombinant LigANI produced in Escherichia coli, are promising vaccine candidates, albeit with variable efficacy. In the present study, LigANI was expressed in the methylotrophic yeast Pichia pastoris using a 12 L bioreactor to produce mannosylated LigANI (mLigANI) for use in a vaccine preparation against leptospirosis. Hamsters immunized with a mLigANI vaccine preparation produced a significant IgG antibody response (P < 0.001) and were protected (83.3 %; P < 0.001) against lethal challenge with 36× LD₅₀ of a virulent strain of L. interrogans serovar Copenhageni. A vaccine preparation based on demannosylated mLigANI (nmLigANI) elicited an immune response in hamsters, but did not afford protection. The production of mLigANI in bioreactor by P. pastoris yielded ~50 mg L^?1 of recombinant protein. P. pastoris is a potential platform for the production of leptospiral antigens on an industrial scale. The results demonstrate that LigANI secreted by P. pastoris on mannosylated form (mLigANI) protect hamsters as subunit vaccine of L. interrogans lethal infection.     

Description of nine South American species of the genus Neralsia cameron, with low scutellar carina (Hymenoptera: Cynipoidea: Figitidae)

In this study a total of nine new species of the genus Neralsia are described for South America: N. desantisi Jiménez & Pujade-Villar n. sp., N. equilatera Jiménez & Pujade-Villar n. sp., N. hermafrodita Jiménez & Pujade-Villar n. sp., N. marioi Jiménez & Pujade-Villar n. sp., N. moisesi Jiménez & Pujade-Villar n. sp., N. obelix Jiménez & Pujade-Villar n. sp., N. parafossulata Jiménez & Pujade-Villar n. sp., N. pseudoneralsia Jiménez & Pujade-Villar n. sp. and N. rauli Jiménez & Pujade-Villar n. sp., all of them have in common the low carina separating the scutellar foveae. Other six known Neralsia species, N. albipennis (Kieffer), N. bogotensis (Kieffer), N. flavidipennis (Kieffer), N. fossulata (Kieffer), N. pilosa (Borgmeier) and N. striaticeps (Kieffer) have the same scutellar character. The morphological characters to differentiate all mentioned species are presented.     

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80–150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 °C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 °C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K_M was 42 mM, and V_max was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.     

Calcium and magnesium binding to human centrin 3 and interaction with target peptides

There are four isoforms of centrin in mammals, with variable sequence, tissue expression, and functional properties. We have recently characterized a number of structural, ion, and target binding properties of human centrin isoform HsCen2. This paper reports a similar characterization of HsCen3, overexpressed in Escherichia coli and purified by phase-reversed chromatography. Equilibrium and dynamic binding studies revealed that HsCen3 has one mixed Ca(2+)/Mg(2+) binding site of high affinity (K(d) = 3 and 10 microM for Ca(2+) and Mg(2+), respectively) and two Ca(2+)-specific sites of low affinity (K(d) = 140 microM). The metal-free protein is fragmented by an unidentified protease into a polypeptide segment of 11 kDa, which was purified by HPLC, and identified by mass spectrometry as the segment of residues 21-112. Similarly, controlled trypsinolysis on Ca(2+)-bound HsCen3 yielded a mixture of segments of residues 1-124 and 1-125. The Ca(2+)/Mg(2+) site could be assigned to this segment and thus resides in the N-terminal half of HsCen3. Temperature denaturation experiments, circular dichroism, and utilization of fluorescence hydrophobic probes allowed us to propose that the metal-free protein has molten globule characteristics and that the dication-bound forms are compact with a polar surface for the Mg(2+) form and a hydrophobic exposed surface for the Ca(2+) form. Thus, HsCen3 could be classified as a Ca(2+) sensor protein. In addition, it is able to bind strongly to a model target peptide (melittin), as well as to peptides derived from the protein XPC and Kar1p, with a moderate Ca(2+) dependence.     

Infestation of arboreal nests of coatis by triatomine species,vectors of Trypanosoma cruzi,in a large Neotropical wetland

The coati (Nasua nasua, Carnivora) is a medium‐sized mammal common in the Pantanal of Brazil. Unlike most mammals, coatis construct arboreal nests used for resting and reproduction. In this region, the coati is an important host of Trypanosoma cruzi, the causative agent of Chagas disease. There are two possible routes through coatis can be infected by T. cruzi: the oral route or the vectorial route. However, the relative importance of each of these routes in the infection of coatis and its role in the sylvatic cycle of the parasite are unknown. Our objectives were to investigate: (i) whether coati nests were infested by triatomine bugs, (ii) what species were frequent in the nests, (iii) whether the triatomines in nests were infected by T. cruzi, and (iv) what were the food resources of these triatomines. Eight of the 24 nests sampled were infested with triatomines, a total of 37 specimens of at least two species (Rhodnius stali and Triatoma sordida). In one nest, R. stali and T. sordida co‐occurred and both fed on multiple resources, including coatis. This is the first report of triatomines occurring in arboreal nests of coatis. The co‐occurrence of two different genera of triatomine vectors and coatis within the limited space of the coati nests provide multiple opportunities for the exchange of the protozoan parasite through both the vectorial and oral transmission routes.     

Neutrophil paralysis in Plasmodium vivax malaria

Background

The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria.

Materials and Methods

Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30–45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients.

Principal Findings

Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8).

Conclusion

Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.     

Distinct properties of the egress‐related osmiophilic bodies in male and female gametocytes of the rodent malaria parasite Plasmodium berghei

Gametogenesis is the earliest event after uptake of malaria parasites by the mosquito vector, with a decisive impact on colonization of the mosquito midgut. This process is triggered by a drop in temperature and contact with mosquito molecules. In a few minutes, male and female gametocytes escape from the host erythrocyte by rupturing the parasitophorous vacuole and the erythrocyte membranes. Electron‐dense, oval‐shaped organelles, the osmiophilic bodies (OB), have been implicated in the egress of female gametocytes. By comparative electron microscopy and electron tomography analyses combined with immunolocalization experiments, we here define the morphological features distinctive of male secretory organelles, hereafter named MOB (male osmiophilic bodies). These organelles appear as club‐shaped, electron‐dense vesicles, smaller than female OB. We found that a drop in temperature triggers MOB clustering, independently of exposure to other stimuli. MDV1/PEG3, a protein associated with OB in Plasmodium berghei females, localizes to both non‐clustered and clustered MOB, suggesting that clustering precedes vesicle discharge. A P. berghei mutant lacking the OB‐resident female‐specific protein Pbg377 displays a dramatic reduction in size of the OB, accompanied by a delay in female gamete egress efficiency, while female gamete fertility is not affected. Immunolocalization experiments indicated that MDV1/PEG3 is still recruited to OB‐remnant structures.