Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.     

Infants' Peripheral Blood Lymphocyte Composition Reflects Both Maternal and Post-Natal Infection with Plasmodium falciparum

Maternal parasitoses modulate fetal immune development, manifesting as altered cellular immunological activity in cord blood that may be linked to enhanced susceptibility to infections in early life. Plasmodium falciparum typifies such infections, with distinct placental infection-related changes in cord blood exemplified by expanded populations of parasite antigen-specific regulatory T cells. Here we addressed whether such early-onset cellular immunological alterations persist through infancy. Specifically, in order to assess the potential impacts of P. falciparum infections either during pregnancy or during infancy, we quantified lymphocyte subsets in cord blood and in infants'' peripheral blood during the first year of life. The principal age-related changes observed, independent of infection status, concerned decreases in the frequencies of CD4⁺, NK^dim and NKT cells, whilst CD8⁺, Treg and Teff cells'' frequencies increased from birth to 12 months of age. P. falciparum infections present at delivery, but not those earlier in gestation, were associated with increased frequencies of Treg and CD8⁺ T cells but fewer CD4⁺ and NKT cells during infancy, thus accentuating the observed age-related patterns. Overall, P. falciparum infections arising during infancy were associated with a reversal of the trends associated with maternal infection i.e. with more CD4⁺ cells, with fewer Treg and CD8⁺ cells. We conclude that maternal P. falciparum infection at delivery has significant and, in some cases, year-long effects on the composition of infants'' peripheral blood lymphocyte populations. Those effects are superimposed on separate and independent age- as well as infant infection-related alterations that, respectively, either match or run counter to them.     

The Influence of Different Fever Definitions on the Rate of Fever in Neutropenia Diagnosed in Children with Cancer

BackgroundThe temperature limit defining fever (TLDF) is based on scarce evidence. This study aimed to determine the rate of fever in neutropenia (FN) episodes additionally diagnosed by lower versus standard TLDF.MethodsIn a single center using a high TLDF (39.0°C tympanic temperature, Limit_Standard), pediatric patients treated with chemotherapy for cancer were observed prospectively. Results of all temperature measurements and CBCs were recorded. The application of lower TLDFs (Limit_Low; range, 37.5°C to 38.9°C) versus Limit_Standard was simulated in silicon, resulting in three types of FN: simultaneous FN, diagnosed at both limits within 1 hour; earlier FN, diagnosed >1hour earlier at Limit_Low; and additional FN, not diagnosed at Limit_Standard.ResultsIn 39 patients, 8896 temperature measurements and 1873 CBCs were recorded during 289 months of chemotherapy. Virtually applying Limit_Standard resulted in 34 FN diagnoses. The predefined relevantly (≥15%) increased FN rate was reached at Limit_Low 38.4°C, with total 44 FN, 23 simultaneous, 11 earlier, and 10 additional (Poisson rate ratio_{Additional/Standard}, 0.29; 95% lower confidence bound, 0.16). Virtually applying 37.5°C as Limit_Low led to earlier FN diagnosis (median, 4.5 hours; 95% CI, 1.0 to 20.8), and to 53 additional FN diagnosed. In 51 (96%) of them, spontaneous defervescence without specific therapy was observed in reality.ConclusionLower TLDFs led to many additional FN diagnoses, implying overtreatment because spontaneous defervescence was observed in the vast majority. Lower TLDFs led as well to relevantly earlier diagnosis in a minority of FN episodes. The question if the high TLDF is not only efficacious but as well safe remains open.     

Will Passive Protection Save Congo Forests?

Central Africa’s tropical forests are among the world’s largest carbon reserves. Historically, they have experienced low rates of deforestation. Pressures to clear land are increasing due to development of infrastructure and livelihoods, foreign investment in agriculture, and shifting land use management, particularly in the Democratic Republic of Congo (DRC). The DRC contains the greatest area of intact African forests. These store approximately 22 billion tons of carbon in aboveground live biomass, yet only 10% are protected. Can the status quo of passive protection — forest management that is low or nonexistent — ensure the preservation of this forest and its carbon? We have developed the SimCongo model to simulate changes in land cover and land use based on theorized policy scenarios from 2010 to 2050. Three scenarios were examined: the first (Historical Trends) assumes passive forest protection; the next (Conservation) posits active protection of forests and activation of the national REDD+ action plan, and the last (Agricultural Development) assumes increased agricultural activities in forested land with concomitant increased deforestation. SimCongo is a cellular automata model based on Bayesian statistical methods tailored for the DRC, built with the Dinamica-EGO platform. The model is parameterized and validated with deforestation observations from the past and runs the scenarios from 2010 through 2050 with a yearly time step. We estimate the Historical Trends trajectory will result in average emissions of 139 million t CO₂ year^-1 by the 2040s, a 15% increase over current emissions. The Conservation scenario would result in 58% less clearing than Historical Trends and would conserve carbon-dense forest and woodland savanna areas. The Agricultural Development scenario leads to emissions of 212 million t CO₂ year^-1 by the 2040s. These scenarios are heuristic examples of policy’s influence on forest conservation and carbon storage. Our results suggest that 1) passive protection of the DRC’s forest and woodland savanna is insufficient to reduce deforestation; and 2): enactment of a REDD+ plan or similar conservation measure is needed to actively protect Congo forests, their unique ecology, and their important role in the global carbon cycle.     

Urinary Calcium and Oxalate Excretion in Healthy Adult Cats Are Not Affected by Increasing Dietary Levels of Bone Meal in a Canned Diet

This study aimed to investigate the impact of dietary calcium (Ca) and phosphorus (P), derived from bone meal, on the feline urine composition and the urinary pH, allowing a risk assessment for the formation of calcium oxalate (CaOx) uroliths in cats. Eight healthy adult cats received 3 canned diets, containing 12.2 (A), 18.5 (B) and 27.0 g Ca/kg dry matter (C) and 16.1 (A), 17.6 (B) and 21.1 g P/kg dry matter (C). Each diet was fed over 17 days. After a 7 dayś adaptation period, urine and faeces were collected over 2×4 days (with a two-day rest between), and blood samples were taken. Urinary and faecal minerals, urinary oxalate (Ox), the urinary pH and the concentrations of serum Ca, phosphate and parathyroid hormone (PTH) were analyzed. Moreover, the urine was microscopically examined for CaOx uroliths. The results demonstrated that increasing levels of dietary Ca led to decreased serum PTH and Ca and increased faecal Ca and P concentrations, but did not affect the urinary Ca or Ox concentrations or the urinary fasting pH. The urinary postprandial pH slightly increased when the diet C was compared to the diet B. No CaOx crystals were detected in the urine of the cats. In conclusion, urinary Ca excretion in cats seems to be widely independent of the dietary Ca levels when Ca is added as bone meal to a typical canned diet, implicating that raw materials with higher contents of bones are of subordinate importance as risk factors for the formation of urinary CaOx crystals.     

Long-term study (1974-1998) of seasonal changes in the phytoplankton in Lake Geneva: a multi-table approach

The monitoring of Lake Geneva began after one decade of eutrophicationand has provided a uniform set of phytoplankton data. This studyaimed to define the mean annual pattern of the seasonal successionsof phytoplankton species, and to determine whether inter-annualdistortions in this seasonal structure occur. We analysed the25 annual patterns using the STATIS multi-table method. Thephytoplankton successions in the first part of the year fittedwell with the pattern predicted by the Plankton Ecology Group(PEG) model. But the temporal evolution in the summer phytoplanktoncommunity differed from the PEG model, and was subject to between-yearsdifferences. We identified three homogeneous periods for theannual patterns: 1974–1985, 1986–1991 (except 1988)and 1992–1996 (including 1988). During the first period,phytoplankton succession followed the reference annual pattern,the typical autumnal community was missing during the secondperiod, and this autumn community developed earlier during thethird period. This study reflects the good ability of the phytoplanktoncommunity of Lake Geneva to resist both inter-annual fluctuationsand brutal shifts in the annual functioning of the system. Webelieve these shifts resulted from a gradual change in environmentalparameters, including the deepening of the phosphorus-depletedlayer and a change in its timing as a result of the long-termmeteorological changes.     

Cellular and subcellular expression of Golf/Gs and Gq/G11 alpha-subunits in rat pancreatic endocrine cells.

We studied the cellular and subcellular localization of Galpha-subunits in pancreas by immunocytochemistry. Golfalpha and G11alpha were specifically localized in islet insulin B-cells and glucagon A-cells, respectively. Gsalpha and Gqalpha labeling was more abundant in B-cells. The presence of Golfalpha in B-cells was confirmed by in situ hybridization. In B-cells, Golfalpha and Gsalpha were found in the Golgi apparatus, plasma membrane (PM) and, remarkably, in mature and immature insulin secretory granules, mainly at the periphery of the insulin grains. Gqalpha was detected on the rough endoplasmic reticulum (RER) near the Golgi apparatus. In A-cells, the Galpha-subunits were mostly within the glucagon granules: G11alpha gave the strongest signal, Gsalpha less strong, Gq was scarce, and Golf was practically absent. Gqalpha and Gsalpha immunoreactivity was detected in acinar cells, although it was much weaker than that in islet cells. The cell-dependent distribution of the Galpha-subunits indicates that the stimulatory pathways for pancreatic function differ in acinar and in islet B- and A-cells. Furthermore, the G-protein subunits in islet cell secretory granules might be functional and participate in granule trafficking and hormone secretion.     

Novel carbocyclic nucleoside analogs suppress glomerular mesangial cells proliferation and matrix protein accumulation through ROS-dependent mechanism in the diabetic milieu

The synthesis of a series of novel 3,4-cis- and 3,4-trans-substituted carbocyclic nucleoside analogs from protected uracil and thymine is described. The key reaction in the followed synthetic protocols utilized the Mitsunobu reaction to couple 3,4-substituted cyclopentanols to ³N-benzoyl uracil or ³N-benzoyl thymine. These molecules were evaluated with regard to their ability to treat diabetic nephropathy. Our results show that two analogs significantly reduced high-glucose induced glomerular mesangial cells proliferation and matrix protein accumulation in vitro and, more interestingly, exhibited an anti-oxidative effect suggesting that the activity may be mediated through ROS-dependent mechanism.