Plasma Free Hemoglobin and Microcirculatory Response to Fresh or Old Blood Transfusions in Sepsis

BackgroundFree hemoglobin (fHb) may induce vasoconstriction by scavenging nitric oxide. It may increase in older blood units due to storage lesions. This study evaluated whether old red blood cell transfusion increases plasma fHb in sepsis and how the microvascular response may be affected.MethodsThis is a secondary analysis of a randomized study. Twenty adult septic patients received either fresh or old (<10 or >15 days storage, respectively) RBC transfusions. fHb was measured in RBC units and in the plasma before and 1 hour after transfusion. Simultaneously, the sublingual microcirculation was assessed with sidestream-dark field imaging. The perfused boundary region was calculated as an index of glycocalyx damage. Tissue oxygen saturation (StO₂) and Hb index (THI) were measured with near-infrared spectroscopy and a vascular occlusion test was performed.ResultsSimilar fHb levels were found in the supernatant of fresh and old RBC units. Despite this, plasma fHb increased in the old RBC group after transfusion (from 0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369] mg/mL, p = 0.006). The sublingual microcirculation was unaltered in both groups, while THI increased. The change in plasma fHb was inversely correlated with the changes in total vessel density (r = -0.57 [95% confidence interval -0.82, -0.16], p = 0.008), De Backer score (r = -0.63 [95% confidence interval -0.84, -0.25], p = 0.003) and THI (r = -0.72 [95% confidence interval -0.88, -0.39], p = 0.0003).ConclusionsOld RBC transfusion was associated with an increase in plasma fHb in septic patients. Increasing plasma fHb levels were associated with decreased microvascular density.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01584999","term_id":"NCT01584999"}}NCT01584999     

Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing

RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.     

Reliable and Accurate CD4+ T Cell Count and Percent by the Portable Flow Cytometer CyFlow MiniPOC and “CD4 Easy Count Kit-Dry”, as Revealed by the Comparison with the Gold Standard Dual Platform Technology

Background

An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the “gold standard” for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the “CD4% Count Kit-Dry”.

Materials and Methods

Venous blood from 59 adult HIV+ patients (age: 25–58 years; 43 males and 16 females) was collected and stained with the “MiniPOC CD4% Count Kit-Dry”. CD4+ count and percentage were then determined in triplicate by the CyFlow MiniPOC. In parallel, CD4 count was performed using mAbs and a CyFlow Counter, or by a dual platform system (from Beckman Coulter) based upon Cytomic FC500 (“Cytostat tetrachrome kit” for mAbs) and Coulter HmX Hematology Analyzer (for absolute cell count).

Results

The accuracy of CyFlow MiniPOC against Cytomic FC500 showed a correlation coefficient (CC) of 0.98 and 0.97 for CD4+ count and percentage, respectively. The accuracy of CyFlow MiniPOC against CyFlow Counter showed a CC of 0.99 and 0.99 for CD4 T cell count and percentage, respectively. CyFlow MiniPOC showed an excellent repeatability: CD4+ cell count and percentage were analyzed on two instruments, with an intra-assay precision below ±5% deviation. Finally, there was no carry-over contamination for samples at all CD4 values, regardless of their position in the sequence of analysis.

Conclusion

The cost-effective CyFlow MiniPOC produces rapid, reliable and accurate results that are fully comparable with those from highly expensive dual platform systems.     

Trace Elements in Scalp Hair Samples from Patients with Relapsing-Remitting Multiple Sclerosis

Background

Epidemiological studies have suggested a possible role of trace elements (TE) in the etiology of several neurological diseases including Multiple Sclerosis (MS). Hair analysis provides an easy tool to quantify TE in human subjects, including patients with neurodegenerative diseases.

Objective

To compare TE levels in scalp hair from patients with MS and healthy controls from the same geographic area (Sicily).

Methods

ICP-MS was used to determine the concentrations of 21 elements (Ag, Al, As, Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Mo, Ni, Pb, Rb, Sb, Se, Sr, U, V and Zn) in scalp hair of 48 patients with relapsing–remitting Multiple Sclerosis compared with 51 healthy controls.

Results

MS patients showed a significantly lower hair concentration of aluminum and rubidium (median values: Al = 3.76 μg/g vs. 4.49 μg/g and Rb = 0.007 μg/g vs. 0.01 μg/g;) and higher hair concentration of U (median values U: 0.014 μg/g vs. 0.007 μg/g) compared to healthy controls. The percentages of MS patients showing hair elemental concentrations greater than the 95th percentile of controls were 20% for Ni, 19% for Ba and U, and 15% for Ag, Mo and Se. Conversely, the percentages of MS patients showing hair elemental concentrations lower than the 5th percentile of healthy controls were 27% for Al, 25% for Rb, 22% for Ag, 19% for Fe, and 16% for Pb. No significant association was found between levels of each TE and age, disease duration or Expanded Disability Status Scale (EDSS) score. After stratification by gender, healthy subjects did not show any significant difference in trace element levels, while MS patients showed significant differences (p<0.01) for the concentrations of Ag, Cr, Fe, Ni and Sr. No significant differences were also found, at p<0.01, in relation to the use of cigarettes, consume of water, vegetables and place of living.

Conclusion

The different distributions of TE in hair of MS patients compared to controls provides an additional indirect evidence of metabolic imbalance of chemical elements in the pathogenesis of this disease. The increase in U and decrease in Al and Rb levels in MS compared to controls require further assessments as well as the observed different distributions of other elements.     

Extracellular Superoxide Dismutase Regulates the Expression of Small GTPase Regulatory Proteins GEFs,GAPs, and GDI

Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3–induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3–driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme.     

Low Dose Infliximab for Prevention of Postoperative Recurrence of Crohn’s Disease: Long Term Follow-Up and Impact of Infliximab Trough Levels and Antibodies to Infliximab

Objective

In patients with postoperative recurrence of Crohn’s disease endoscopic and clinical remission can be maintained for up to 1 year with low infliximab doses (3 mg/Kg). However, in theory low-dose infliximab treated patients could develop subtherapeutic trough levels, infiximab antibodies, and might loose response to therapy. To verify this hypothesis infliximab pharmacokinetics and clinical/endoscopic response were checked in a group of patients treated in the long term with low infliximab doses.

Design

Infliximab antibodies, infliximab levels, highly-sensitive CRP and fecal calprotectin were measured during the 8-week interval in 5 consecutive patients in clinical (Crohn’s Disease Activity Index < 150) and endoscopic (Rutgeerts scores 0–1) remission after one year of therapy with infliximab 3 mg/Kg. For comparison with reported standards, infliximab pharmacokinetics and inflammatory parameters were also tested in 6 Crohn’s disease patients who did not undergo surgery and who were in clinical remission while on infliximab 5 mg/Kg. Patients on low infliximab dose also underwent colonoscopy after 18 additional months of therapy.

Results

Highly sensitive CRP and fecal calprotectin increased in all patients during the 8-week interval. Infliximab trough levels were lower in patients treated with the low dose compared to controls (mean±SE: 2.0±0.3 vs 4.75±0.83 μg/mL respectively p<0.05). Infliximab antibodies were present in two of the subjects treated with low infliximab dose and in none of the controls. However, in low dose-treated patients after 18 additional months of therapy endoscopy continued to show mucosal remission and none of them developed clinical recurrence or side effects.

Conclusions

Patients treated with low infliximab doses had lower trough levels compared to patients treated with 5 mg/Kg and some developed antibodies to infliximab. However, low infliximab doses sustained clinical and endoscopic remission for a total of 30 months of treatment.     

Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis

During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.     

Strange Little Flies in the Big City: Exotic Flower-Breeding Drosophilidae (Diptera) in Urban Los Angeles

Urban landscapes are commonly considered too mundane and corrupted to be biotically interesting. Recent insect surveys employing 29 Malaise traps throughout Los Angeles, California, however, have uncovered breeding populations of two unexpected species of one of the most studied and familiar groups of organisms, Drosophila “fruit” flies. Unlike most introduced species of drosophilids, which breed in fresh or decaying fruits, these are specialized flower-breeders. A common species in the survey was Drosophila (Drosophila) gentica Wheeler and Takada, previously collected only once, in El Salvador. It belongs to the flavopilosa species group, all species of which have been known until now from central Chile, Argentina and Uruguay, to Veracruz, Mexico and the Caribbean, breeding in flowers of Cestrum (“jessamine”) and Sessea (Solanaceae). The Los Angeles populations are probably breeding in a native and/or introduced Cestrum; in addition, populations in San Luis Obispo County were visiting ornamental Cestrum. Drosophila gentica occurs as far north as San Francisco, where it was found breeding in Cestrum aurantiacum. D. gentica is redescribed and figured in detail for diagnostic and identification purposes. Specimens from Jamaica previously identified as D. gentica are a distinct species but are not formally described in lieu of complete male specimens. Rare in the Malaise traps was Drosophila (Sophophora) flavohirta Malloch, a common species in Australia on the blossoms of native Myrtaceae, found on introduced Eucalyptus in South Africa and both Eucalyptus and Syzygium in Madagascar; adults feed on myrtaceous pollen and nectar, larvae breed in the flowers. It is also redescribed in detail, including its unusual egg. This is the first New World report of this species; DNA sequences confirm it is a morphologically highly aberrant member of the D. melanogaster species group. This study reveals how intensive field sampling can uncover remarkable biodiversity in even the most urbanized areas.