Cytochemical and electrophoretic studies of haemoglobin synthesis in the fat body of a midge, Chironomus thummi

The fat body of developing mid- and late fourth instar larvae of a midge, Chironomus thummi, has been investigated by means of the benzidine reaction for the localization of haemoglobin within cells. In the subepidermal fat body the reaction deposits of the haemoglobin pseudo-peroxidase activity appear predominantly in the intracisternal cavities of ER and the Golgi, and later, in the pharate pupal stage, in small dense granules (0.5–1 μm in. diameter).All the major protein bands of fat body extracts, which are resolved in electrophoresis, give the benzidine reaction and show incorporation of ¹⁴C-amino levulinic acids, in this case a specific marker for haemoglobin synthesis. In addition, labelled proteins show identical electrophoretic mobility as the haemoglobins of the haemolymph, suggesting that haemoglobins are synthesized in the fat body. Two types of fat body cells seem to differ with respect to their rôle in haemoglobin metabolism.     

Fat body: a site of hemoglobin synthesis in Chironomus thummi (diptera)

Fourth instar larvae of Chironomus thummi were permitted to incorporate labeled amino acids and/or sigma-aminolevulinic acid (sigma-ALA) in vivo and in organ culture. The products secreted into the hemolymph or into the culture medium were examined by acrylamide gel electrophoresis. Nine electrophoretic bands can be resolved as hemoglobins without staining. When gels are sliced for scintillation counting, incorporated amino acids and sigma-ALA are shown to be associated primarily with the same nine hemoglobin bands, suggesting that hemoglobins are assembled and secreted. Staining of gels with Coomassie brilliant blue reveals that there are several bands in addition to the visible hemoglobins. These bands incorporate amino acids, but not sigma-ALA, suggesting that they are non-heme proteins. The results of culturing isolated salivary glands, gut, and fat body demonstrate that the fat body is the major site of hemoglobin synthesis and secretion. Labeled products of the gut represent about 5% of the total hemoglobins produced by the tissues, while no hemoglobins are produced by the salivary glands. Although nine hemoglobins are visibly resolved on gels, labeling techniques reveal as many as 14 hemoglobins. This is the first demonstration of hemoglobin synthesis by specific tissues in culture in an invertebrate.     

A New In Vitro Model to Study Cellular Responses after Thermomechanical Damage in Monolayer Cultures

Although electrosurgical instruments are widely used in surgery to cut tissue layers or to achieve hemostasis by coagulation (electrocautery), only little information is available concerning the inflammatory or immune response towards the debris generated. Given the elevated local temperatures required for successful electrocautery, the remaining debris is likely to contain a plethora of compounds entirely novel to the intracorporal setting. A very common in vitro method to study cell migration after mechanical damage is the scratch assay, however, there is no established model for thermomechanical damage to characterise cellular reactions. In this study, we established a new in vitro model to investigate exposure to high temperature in a carefully controlled cell culture system. Heatable thermostat-controlled aluminium stamps were developed to induce local damage in primary human umbilical vein endothelial cells (HUVEC). The thermomechanical damage invoked is reproducibly locally confined, therefore allowing studies, under the same experimental conditions, of cells affected to various degrees as well as of unaffected cells. We show that the unaffected cells surrounding the thermomechanical damage zone are able to migrate into the damaged area, resulting in a complete closure of the ‘wound’ within 48 h. Initial studies have shown that there are significant morphological and biological differences in endothelial cells after thermomechanical damage compared to the mechanical damage inflicted by using the unheated stamp as a control. Accordingly, after thermomechanical damage, cell death as well as cell protection programs were activated. Mononuclear cells adhered in the area adjacent to thermomechanical damage, but not to the zone of mechanical damage. Therefore, our model can help to understand the differences in wound healing during the early phase of regeneration after thermomechanical vs. mechanical damage. Furthermore, this model lends itself to study the response of other cells, thus broadening the range of thermal injuries that can be analysed.     

Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

The important role of the CD8⁺ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8⁺ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8⁺ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4⁺ T-cell set points were observed in PHI subjects with higher HIV-specific CD8⁺ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8⁺ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection.     

Outbreak of Shiga Toxin-Producing Escherichia coli (STEC) O157:H7 Associated with Romaine Lettuce Consumption, 2011

Background

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is the causal agent for more than 96,000 cases of diarrheal illness and 3,200 infection-attributable hospitalizations annually in the United States.

Materials and Methods

We defined a confirmed case as a compatible illness in a person with the outbreak strain during 10/07/2011-11/30/2011. Investigation included hypothesis generation, a case-control study utilizing geographically-matched controls, and a case series investigation. Environmental inspections and tracebacks were conducted.

Results

We identified 58 cases in 10 states; 67% were hospitalized and 6.4% developed hemolytic uremic syndrome. Any romaine consumption was significantly associated with illness (matched Odds Ratio (mOR) = 10.0, 95% Confidence Interval (CI) = 2.1–97.0). Grocery Store Chain A salad bar was significantly associated with illness (mOR = 18.9, 95% CI = 4.5–176.8). Two separate traceback investigations for romaine lettuce converged on Farm A. Case series results indicate that cases (64.9%) were more likely than the FoodNet population (47%) to eat romaine lettuce (p-value = 0.013); 61.3% of cases reported consuming romaine lettuce from the Grocery Store Chain A salad bar.

Conclusions

This multistate outbreak of STEC O157:H7 infections was associated with consumption of romaine lettuce. Traceback analysis determined that a single common lot of romaine lettuce harvested from Farm A was used to supply Grocery Store Chain A and a university campus linked to a case with the outbreak strain. An investigation at Farm A did not identify the source of contamination. Improved ability to trace produce from the growing fields to the point of consumption will allow more timely prevention and control measures to be implemented.     

5-Methoxyleoligin,a Lignan from Edelweiss,Stimulates CYP26B1-Dependent Angiogenesis In Vitro and Induces Arteriogenesis in Infarcted Rat Hearts In Vivo

Background

Insufficient angiogenesis and arteriogenesis in cardiac tissue after myocardial infarction (MI) is a significant factor hampering the functional recovery of the heart. To overcome this problem we screened for compounds capable of stimulating angiogenesis, and herein investigate the most active molecule, 5-Methoxyleoligin (5ML), in detail.

Methods and Results

5ML potently stimulated endothelial tube formation, angiogenic sprouting, and angiogenesis in a chicken chorioallantoic membrane assay. Further, microarray- and knock down- based analyses revealed that 5ML induces angiogenesis by upregulation of CYP26B1. In an in vivo rat MI model 5ML potently increased the number of arterioles in the peri-infarction and infarction area, reduced myocardial muscle loss, and led to a significant increase in LV function (plus 21% 28 days after MI).

Conclusion

The present study shows that 5ML induces CYP26B1-dependent angiogenesis in vitro, and arteriogenesis in vivo. Whether or not CYP26B1 is relevant for in vivo arteriogenesis is not clear at the moment. Importantly, 5ML-induced arteriogenesis in vivo makes the compound even more interesting for a post MI therapy. 5ML may constitute the first low molecular weight compound leading to an improvement of myocardial function after MI.     

Regulation of the epithelial calcium channel TRPV6 by the serum and glucocorticoid-inducible kinase isoforms SGK1 and SGK3

Epithelial calcium (re)absorption is mediated by TRPV5 and TRPV6 channels. TRPV5 is modulated by the SGK1 kinase, a process requiring the PDZ-domain containing scaffold protein NHERF2. The present study explored whether TRPV6 is similarly regulated by SGKs and the scaffold proteins NHERF1/2. In Xenopus oocytes, SGKs activate TRPV6 by increasing its plasma membrane abundance. Deletion of the putative PDZ binding motif on TRPV6 did not abolish channel activation by SGKs. Furthermore, coexpression of neither NHERF1 nor NHERF2 affected TRPV6 or potentiated the SGKs stimulating effect. The present observations disclose a novel TRPV6 regulatory mechanism which presumably participates in calcium homeostasis.