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Why do mosquitoes not collect blood after platelets activate?

Why do mosquitoes not collect blood after platelets activate?



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This may be anecdotal…

The Indian sub-continent is in the rainy part of summer. It follows the mosquito population goes up.

So when I sliced my palm, I expected the mosquitoes to collect there in force. Yet whilst they continued to go about their business on the forearms, face and elsewhere - the blood flowing out of my palm held no attraction for them. This even after I deliberately stood still for nearly a minute! Perhaps the clotting process/platelets/etc were a repellent.

Why do mosquitoes not collect blood after platelets activate?


Blood from a cut or a wound is tainted with bacteria from the surrounding environment. To a mosquito from an evolutionary perspective it would be like eating off the floor. The blood which flows through your veins is clean(er) due to your immune system and so evolution selected a pathway based on insertion into the skin in order to obtain the meal.


Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration

Immune complexes (ICs) form when antibodies encounter their antigens. ICs are present in blood in multiple pathological conditions. Given the abundance of platelets in blood and that they express a receptor for ICs, called Fcγ receptor IIA (FcγRIIA), we examined the impact of ICs in blood in a mouse model. We found that circulating ICs induced systemic shock, characterized by loss of consciousness, by activating platelet FcγRIIA. Shock was mediated by the liberation of serotonin, a molecule better known for its role in the brain, from platelet granules. During shock, platelets were sequestered in the lungs and brain and returned to the blood circulation after their degranulation. Platelets are thus crucial in response to ICs.


Introduction

The recent “International Consensus on (ICON) Anaphylaxis” described anaphylaxis as 𠇊 serious, generalized or systemic, allergic or hypersensitivity reaction that can be life-threatening or fatal”. 1 This definition is intentionally “generic”, in that it doesn't mention any of the specific immune elements that might be involved in particular instances of the disorder, as these may vary depending on individual circumstances. In this review, we will describe the key immune elements, such as antibody isotypes, effector cells, and biological mediators, which can contribute to development and pathophysiological manifestations of anaphylaxis. We in particular will note the extent of evidence implicating these immune components in anaphylaxis in humans versus that induced in mouse models of the disorder, focusing especially on forms of anaphylaxis induced by the reactions of allergens with antigen-specific antibodies. We will not extensively review forms of anaphylaxis induced by the antibody-independent activation of effector cells such as mast cells and basophils, topics which have been reviewed elsewhere. 2, 3


Transfusion-Related Adverse Events

Acute Hemolytic Transfusion Reactions

Acute hemolytic transfusion reactions (AHTRs) occur in 1:76 000 RBC transfusions ( Eder and Chambers, 2007 ). Fatal AHTRs occur in 1:1 800 000 transfusions ( Vamvakas and Blajchman, 2009 ). Typically AHTRs are hemolytic reactions that occur within 24 h of transfusion and lead to intravascular (rarely extravascular) hemolysis secondary to ABO incompatibility, but may result from other blood group incompatibilities. In contrast, DHTRs typically result in extravascular hemolysis.

Intravascular hemolysis is caused by RBC antigen interaction with preformed antibodies. Most often these are ABO (IgM) antibodies however, sufficient concentrations of preformed IgG can also play a role. Other blood group incompatibilities can result in AHTR, such as antibodies to Kidd or Duffy blood group system antigens. Finally, incompatible plasma may be transfused to a patient and result in an AHTR. Most frequently, this occurs secondary to ABO incompatible platelet transfusion. The degree of hemolysis depends on the amount and strength (titer) of the ABO antibody.

After the initial antigen–antibody interaction, complement activation occurs, leading to formation of the membrane attack complex and eventual destruction of the RBC with the release of its components into the blood stream. Initially, hemoglobin is bound by haptoglobin and metabolized. When haptoglobin stores are exhausted, the hemoglobin and other constituents, such as RBC stroma, may travel to the kidney, leading to tissue damage due to toxicity. In addition, the RBC constituents can activate the coagulation cascade and lead to systemic vasoconstriction and shock ( Bellone and Hillyer, 2013 ).

AHTRs are characterized by fever, back or flank pain, chest pain, hypotension, shock, and ‘impending sense of doom’ in the patient. These findings often occur soon after the start of the transfusion. The transfusion should be stopped immediately treatment includes vigorous hydration, furosemide (to maintain urine output), as well as support of blood pressure, disseminated intravascular coagulation, and other clinical sequelae ( Ludens et al., 1968 ).

A transfusion reaction workup should be initiated quickly to identify the cause of the reaction. Blood bank testing may reveal hemolysis, incompatibility between the donor and recipient, and positive DAT. Other laboratory analysis that may be helpful includes testing of bilirubin and LDH levels, and urinalysis (to identify free hemoglobin in the urine).

Human error is the most common cause of AHTRs due to ABO incompatibility. The error could be made in many places: during the initial blood draw, issuing of the blood product, and transfusing product to the wrong patient. When an AHTR occurs, a root cause analysis is imperative (and mandated by The Joint Commission) to initiate corrective action and prevent such occurrences in the future ( Bellone and Hillyer, 2013 ). Efforts to further decrease the human error component include computerized matching of patient armband barcodes to blood product barcodes. Prevention mandates elimination of the possibility of human error. In contrast, due to low platelet inventories and short shelf-life of platelets, ABO incompatible platelet transfusions occur not uncommonly. AHTRs secondary to platelet transfusions are mitigated by transfusing low titer platelets or ABO compatible platelets.


Discussion

The presence of salivary ADA activity in hematophagous insects is puzzling, because adenosine is a vasodilatory and anti-platelet substance (Collis, 1989 Dionisotti et al., 1992 Chinellato et al., 1994), which might be expected to increase blood availability at the feeding site. However, adenosine may be implicated in peripheral pain perception (Burnstock and Wood, 1996 Poon and Sawynok, 1999 Charlab et al., 2000), although its role in nociception is contradictory: while adenosine A2 and A3 receptors appear to be clearly nociceptive, adenosine A1 receptor stimulation appears to be anti-nociceptive (Poon and Sawynok, 1998 Poon and Sawynok, 1999). Another effect of salivary ADA may be related to a local accumulation of inosine at the feeding site. Inosine has been shown to be a potent inhibitor of the production of the inflammatory cytokines TNF-α, I l -1, I l -12, macrophage-inflammatory protein-1α and IFN-γ (Hasko et al., 2000). I l -1, in particular, is a potent peripheral nociceptive agent (Bianchi et al., 1998). However, it is doubtful whether these cytokines could be produced by the host and released during the few minutes that mosquitoes take to feed, although they may have an effect on the fate of the host immune response to salivary allergens and pathogens co-injected with the mosquito saliva. While the role of adenosine in nociception is unclear, adenosine is known to induce mast cell degranulation (Tilley et al., 2000), resulting in the release of histamine. From the point of view of the mosquito, the itching induced by histamine would have the undesirable effect of alerting the host to its presence. Although it is clear that adenosine could be beneficial to a hematophagous insect, to the point that phlebotomine sand flies produce copious amounts of the substance in their salivary glands (Ribeiro et al., 1999 Ribeiro and Modi, 2001), the presence of ADA in the saliva of other hematophagous insects indicates that, if it has any evolutionary significance, adenosine may not be a desirable substance at the feeding site.

In accordance with the ambivalent role of adenosine in blood feeding, the data presented in this paper indicate that the presence of salivary ADA in mosquitoes is not a consistent finding, unlike the ubiquitous occurrence of diverse anticoagulants or the enzyme apyrase (Ribeiro, 1995). No ADA activity was found in Anopheles gambiae, but relatively high activities were found in both Culex quinquefasciatus and Aedes aegypti. The specific activity of 16munitsglandpair −1 for Aedes aegypti represents a specific activity of 5unitsmg −1 salivaryprotein, while the value of 2.6munitsglandpair −1 for C. quinquefasciatus represents 2.5unitsmg −1 salivaryprotein. This amount is larger than the specific activity of erythrocyte ADA (0.2unitsmg −1 protein Agarwal et al., 1975) and is similar to that in tissues specialized in digestion, such as the crude ADA sold by Sigma Chemical Co. obtained from calf intestinal mucosa (1–5unitsmg −1 protein) (Sigma Chemical catalog, 2000–2001 edition). In the case of Aedes aegypti, salivary ADA appears to be secreted during a blood meal, as demonstrated by the significant decrease in levels in the salivary glands following a blood meal, its presence in solutions probed by hungry female mosquitoes and its presence in serotonin-induced saliva. In C. quinquefasciatus, no activity could be detected in serotonin-induced saliva, although very low levels of the enzyme (less than 0.03% of the content of one pair of glands per probing mosquito) were left in feeders probed by this species. No significant decrease in enzyme levels was detected in C. quinquefasciatus salivary glands following a blood meal. This suggests that ADA serves a largely endogenous function in the salivary glands of C. quinquefasciatus.

We can only speculate about the evolutionary scenario that has led to the presence or absence of ADA in the saliva of hematophagous arthropods, but we may assume that adenosine has both positive and negative effects on blood feeding. The positive effects could be due to its actions on platelet aggregation and vasodilation. Perhaps for this reason, the sand flies Phlebotomus papatasi and P. argentipes have abundant adenosine in their salivary glands (Ribeiro et al., 1999 Ribeiro and Modi, 2001). It is possible that these phlebotomine hosts are less sensitive to the negative effects of adenosine, that they feed when their hosts are in profound sleep and unresponsive to relatively small disturbances or that sand flies have other mechanisms to counteract the negative effects of adenosine on feeding. However, both Aedes aegypti and C. quinquefasciatus have their own salivary vasodilatory molecules: the peptide sialokinin in Aedes aegypti (Champagne and Ribeiro, 1994) and an unknown substance in C. quinquefasciatus (Ribeiro, 2000). In addition, Aedes aegypti has large amounts of salivary apyrase, which inhibits platelet aggregation (Ribeiro et al., 1984b), while C. quinquefasciatus has a potent salivary phospholipase C that destroys platelet activating factor (J. M. C. Ribeiro and I. Francischetti, in preparation). Other molecules can therefore replace the positive functions of adenosine at the feeding site, and we are left only with its negative effects.

If we assume that the negative role of adenosine is related to its ability to bring the attention of the host to the feeding site, then the intensity of this negative effect would be affected by two variables: the degree to which adenosine induces pain or itching at the feeding site and the behavioral response of the host to pain and itching. The first variable can vary from species to species, e.g. the density of mast cells in the skin of the host selected for feeding by the mosquito, the number and affinity of adenosine receptors inducing cell degranulation or the density of adenosine nociceptive receptors in the skin. The second variable depends, for example, on whether the vertebrate is asleep or alert and how it reacts to the same stimulus in these two conditions. Depending on these variables, it may be of more or less selective advantage to neutralize the negative effect of adenosine on blood feeding, and more than one strategy may be employed to counteract its effects. Thus, adenosine could be destroyed via ADA or its effects reduced by the use of physiological inhibitors, e.g. molecules that act in an opposite way on the target cell, such as mast cell degranulation inhibitors or anesthetics.

It is interesting to note that Aedes aegypti is a diurnal feeder and that its main hosts are humans. Salivary homogenates from Aedes aegypti have the ability to prevent the release of tumor necrosis factor (TNF) by mast cells (Bissonette et al., 1993), indicating that this insect may be under pressure to avoid causing degranulation of the mast cells of the host. In contrast, C. quinquefasciatus is a nocturnal feeder on humans and may avoid the negative behavioral effects of adenosine by feeding when the physiological state of the host is less responsive or unresponsive to an increase in the levels of adenosine at the feeding site. Alternatively, C. quinquefasciatus may have additional salivary components that antagonize the nociceptive and/or mast cell degranulating effects of adenosine. In this regard, it is interesting to note that both triatomine bugs (Ribeiro and Walker, 1994) and ticks (Paesen et al., 1999) have salivary proteins that neutralize histamine, and an anesthetic substance has recently been reported from the saliva of a triatomine bug (Dan et al., 1999). The presence of ADA in Aedes aegypti may represent one of several ways in which the negative effects of adenosine can be counteracted. While these considerations may help to explain why Aedes aegypti could benefit from secreting ADA, we have no clue as to the role of salivary ADA in C. quinquefasciatus, although we can speculate that it may be involved in some endogenous function, such as regulating transmitter signaling by deactivating adenosine or producing inosine or an inosine derivative as a secretory product. Such speculations suggest the hypothesis that anti-nociceptive and anti-mast cell salivary substances should be found abundantly in the salivary glands of hematophagous arthropods.

From a biochemical perspective, it is interesting that the salivary AMP deaminase activity of C. quinquefasciatus is approximately three times greater than that of ADA, while in Aedes aegypti it is one-tenth of the ADA activity. AMP deaminase and ADA are enzymes that probably evolved by gene duplications (Becerra and Lazcano, 1998). Assuming that the two activities reside in a single enzyme in mosquito salivary glands, cloning and expression of these two enzymes, one from each mosquito species, might indicate the amino acid residues that confer greater specificity towards either AMP or adenosine. Alternatively, each mosquito species may express the two enzymes (ADA and AMP deaminase) in their glands in different ratios.

The cDNA discussed in this paper may or may not be responsible for the majority of the enzyme activity seen in C. quinquefasciatus salivary glands. The work reported here confirms that sequencing cDNA libraries from salivary glands of hematophagous insects can yield information leading to the detection of many salivary enzymes not previously identified, such as amylases, hyaluronidases, ADA and 5′-nucleotidases (Charlab et al., 1999 Charlab et al., 2000). Coupled with appropriate biochemical and pharmacological assays, this approach lends impetus to our understanding of how insects have evolved to become one of the most successful and diverse groups of species in the animal kingdom.


STEMI: Management

P2Y12 Inhibitors

The P2Y12 inhibitors are useful for further inhibiting platelet activation and aggregation. The choice of the P2Y12 receptor blocker (Clopidogrel, Prasugrel, and Ticagrelor) depends on the reperfusion strategy.

Clopidogrel ( Table 2 ) is a second-generation thienopyridine that irreversibly antagonizes the platelet P2Y12 ADP receptor. Clopidogrel added to aspirin reduces the risk of death and other cardiovascular events in who will be treated with fibrinolysis ( Steg et al., 2012 Chen et al., 2005a Sabatine et al., 2005 ). However, in the setting of primary PCI, current guidelines prefer ticagrelor or prasugrel, because they have been proven to be superior to clopidogrel in large outcome trials in the risk of death and other major cardiovascular events at 1-year follow-up ( Wiviott et al., 2007 Wallentin et al., 2009 O’Gara et al., 2013 Steg et al., 2012 ).

Prasugrel ( Table 2 ) is a third-generation thienopyridine P2Y12 inhibitor, which is metabolized to its active form more quickly and fully than clopidogrel, and thus has a more potent and consistent effect ( Gurbel et al., 2012 ). The TRITON trial compared the use of prasugrel versus clopidogrel in patients with acute coronary syndromes receiving aspirin. The study showed that the use of prasugrel was associated with a reduction in risk of cardiovascular death, nonfatal myocardial infarction, and nonfatal stroke, but an increased risk of major bleeding ( Wiviott et al., 2007 ). Therefore, prasugrel is contraindicated in patients with prior stroke/transient ischemic attack or active pathological bleeding. Lower body weight (< 60 kg) and age ≥ 75 years are relative contraindications ( Steg et al., 2012 ).

Ticagrelor ( Table 2 ) is a new P2Y12 receptor blocker. Unlike clopidogrel or prasugrel, ticagrelor is not a thienopyridine. It is a direct-acting agent with a reversible effect, which does not require biotransformation and binds reversibly to the P2Y12 receptors, antagonizing ADP signaling and platelet activation. The PLATO trial compared ticagrelor to clopidogrel in patients with acute coronary syndromes treated with aspirin and other standard therapy. Results showed that ticagrelor reduces the composite of cardiovascular death, myocardial infarction, or stroke at 12 months, without an increase in the rate of overall major bleeding, but with an increase in the rate of non-procedure-related bleeding ( Wallentin et al., 2009 ).


Conclusions

Although further examination regarding the pathogen infection in the flightless mosquitoes remains to be elucidated, the mosquito rearing method for Aedes species presented here was extremely secure, and the protocol was inexpensive and less labor intensive, making it possible to perform intensive studies using mosquitoes carrying human hazardous pathogens, even in counties where highly secure containment systems are mandatory. This protocol could also be applicable to Anopheles and Culex mosquitoes, which are major vectors for other devastating diseases, such as malaria, West Nile fever, and Japanese encephalitis.


Platelet-Rich Plasma: The Choice of Activation Method Affects the Release of Bioactive Molecules

Platelet-Rich Plasma (PRP) is a low-cost procedure to deliver high concentrations of autologous growth factors (GFs). Platelet activation is a crucial step that might influence the availability of bioactive molecules and therefore tissue healing. Activation of PRP from ten voluntary healthy males was performed by adding 10% of CaCl2, 10% of autologous thrombin, 10% of a mixture of CaCl2 + thrombin, and 10% of collagen type I. Blood derivatives were incubated for 15 and 30 minutes and 1, 2, and 24 hours and samples were evaluated for the release of VEGF, TGF-β1, PDGF-AB, IL-1β, and TNF-α. PRP activated with CaCl2, thrombin, and CaCl2/thrombin formed clots detected from the 15-minute evaluation, whereas in collagen-type-I-activated samples no clot formation was noticed. Collagen type I produced an overall lower GF release. Thrombin, CaCl2/thrombin, and collagen type I activated PRPs showed an immediate release of PDGF and TGF-

that remained stable over time, whereas VEGF showed an increasing trend from 15 minutes up to 24 hours. CaCl2 induced a progressive release of GFs from 15 minutes and increasing up to 24 hours. The method chosen to activate PRP influences both its physical form and the releasate in terms of GF amount and release kinetic.

1. Introduction

Tissue repair in musculoskeletal injuries is often a slow and sometimes incomplete process, with patient suffering pain and limited function, and therefore it is accompanied by high costs to society, in terms of both money spent on healthcare and also loss of work. Thus, many efforts have been made in order to investigate new approaches to increase the regenerative potential and favour tissue healing. Since several studies have underlined the role of growth factors (GFs) in the regulation of normal tissue structure and the reaction to tissue damage, their use is thought to be useful in clinical practice to promote rapid healing with high quality tissue and allow an early and safe return to unrestricted activity [1].

Platelets constitute a reservoir of critical GFs and cytokines which may govern and regulate the tissue healing process. The bioactive molecules secreted by platelet α-granules are involved in several cellular activities such as stem cell trafficking, proliferation, and differentiation, with a complex effect on pro/anti-inflammatory and anabolic/catabolic processes [2]. Moreover, with respect to purified individual GFs, platelets have the theoretical advantage of containing various bioactive molecules with a natural balance of anabolic and catabolic functions, possibly optimizing the tissue environment and favouring the healing process [2]. Based on this rationale, Platelet-Rich Plasma (PRP) is an easy, low-cost, and minimally invasive procedure to deliver high concentrations of autologous GFs and cytokines into injured tissues in physiological proportions. This blood-derived product, placed directly into the damaged tissue, either surgically or through injections, has been widely experimented in different fields of medicine [3–10].

However, despite the numerous benefits ascribed to PRP and the promising results reported for its therapeutic potential, the clinical outcomes are heterogeneous and sometimes contradictory. These controversial findings are due to both the different clinical protocols applied, making it difficult to compare results and draw conclusions about its real efficacy, and even more so the lack of standardization in PRP preparation procedures. This has led to the availability of a huge number of products differing in terms of cell types and quantity and therefore GF and cytokine content and release times. Among the several variables affecting PRP releasate, platelet activation is a crucial step that might influence the availability of bioactive molecules and therefore tissue healing [10, 11].

The term “activation” refers to 2 key processes that are initiated during PRP preparation: (1) degranulation of platelets to release GFs from α-granules and (2) fibrinogen cleavage to initiate matrix formation, a clotting process which allows the formation of a platelet gel, and therefore to confine the secretion of molecules to the chosen site [12]. An activation step before PRP administration is included in many of the protocols used, commonly by adding thrombin and/or calcium chloride (CaCl2), but some physicians prefer to inject PRP in its resting form, relying on the spontaneous platelet activation occurring after exposure to the native collagen present in the connective tissues [13]. Currently, there is a lack of evidence on the most suitable method for PRP activation, and the choice of strategy to activate it is mainly based on practical reasons rather than supported by studies on the effects of the different procedures on the final platelet releasate. The definition of the differences among activation methods might allow PRP preparations to be optimized, by identifying the most suitable strategy for each specific pathology, in order to obtain a customized PRP for the various clinical indications.

The aim of the present study is therefore to compare different strategies to activate PRP, by evaluating the content of both GFs and cytokines, as well as their release kinetics.

2. Materials and Methods

This study was approved by the local Ethics Committee and the Institutional Review Board, and each donor signed an informed written consent. PRP, Platelet-Poor Plasma (PPP), and autologous thrombin were obtained from ten voluntary healthy men (mean age ± SD:

years) who underwent a blood sample collection of 150 mL. Subjects did not present with systemic disorders, smoking habit, infection, nonsteroidal anti-inflammatory drug use in the 5 days before blood donation, haemoglobin lower than 11 g/dL, or platelets lower than 150 × 10 3 /μL. A code number was assigned to each sample to ensure the subject’s anonymity.

2.1. Preparation and Activation of Blood Derivatives

PRP, PPP, and autologous thrombin were prepared by a whole blood separator (Angel, Cytomedix Inc., Gaithersburg, MD). For PRP preparation, 150 mL of venous blood was drawn from each donor and transferred into an Angel centrifuge chamber and centrifuged for 25 min at two different speeds: at 3500 rpm for the first 3 minutes and at 3000 rpm for the remaining time. Then, PRP was extracted from the buffy-coat into an empty sterile syringe. PPP was collected from another bag and transferred into a new syringe. Autologous thrombin was prepared starting from PPP according to the manufacturer’s instructions.

2.2. Platelet Concentration and White Blood Cell Number

The platelet concentration and the white blood cell (WBC) number of PRP, PPP, and peripheral blood (PB) were analysed with an automated blood cell counter (COULTER LH 750 Haematology Analyzer Beckman Coulter SRL, Milan, Italy). Linearity was 5–1000 × 10 3 /μL for platelet count and 0.1–100 × 10 3 /μL for white blood cell count.

2.3. PRP Activation

Activation of PRP was performed by adding 10% of CaCl2 (final concentration 22.8 mM), 10% of autologous thrombin, 10% of a mixture of CaCl2 + thrombin, and 10% of collagen type I (final concentration 4 μg) (Mascia Brunelli SpA, Milan). PRP without activation and PPP were used as control. Blood derivatives were incubated for 15 and 30 minutes and 1, 2, and 24 hours at 37°C. Then, samples were centrifuged at 2800 ×g for 15 minutes at 20°C, and the supernatants were collected and stored at −80°C until use.

2.4. Growth Factor Evaluation

PRP and PPP were evaluated for the release of VEGF, TGF-β1, PDGF-AB, IL-1β, and TNF-α (R&D Systems, Minneapolis, MN). Samples were thawed at 4°C and centrifuged before analysis then they were assayed in duplicate and factors were evaluated using quantitative sandwich enzyme immunoassays following the manufacturer’s instructions. Results were expressed as pg/mL.

2.5. Statistical Analysis

All continuous normally distributed data were expressed in terms of the mean and the standard deviation of the mean the median was used for not normally distributed ones. The Kolmogorov Smirnov test was performed to test normality of continuous variables. The area under the curve of release at every time of measurement was calculated for each activation method to quantify the amount and kinetics of the released molecules. The Repeated Measures General Linear Model (GLM) with Sidak test for multiple comparisons was performed to assess the differences at different follow-up times in each activation method. The Repeated Measures GLM was also used to assess the influence of the different activation methods.

was considered significant.

All statistical analyses were performed using SPSS v. 19.0 (IBM Corp., Armonk, NY, USA).

3. Results

3.1. Platelet Concentration and White Blood Cell Number

The median number of platelets per cubic millimeter was

for PB, PP, and PRP, respectively. The median concentration of white blood cells per cubic millimeter was

, , and for PB, PP, and PRP, respectively.

3.2. Clot Formation

CaCl2, thrombin, CaCL2/thrombin, and collagen type I induced a different platelet aggregation. In particular, PRP activated with CaCl2, thrombin, and CaCL2/thrombin formed clots detected in the 15-minute evaluation and persisting up to 24 hours (thrombin and CaCL2/thrombin already macroscopically stable at 15 minutes, CaCl2 starting at 15 and visually stabilized at 30 minutes), whereas in collagen-type-I-activated samples no clot formation was noticed for any of the time points evaluated (Figure 1).

3.3. Growth Factor and Cytokine Release

No detectable levels of IL-1β and TNF-α secretion were found in any of the samples at any of the experimental times evaluated.

Significantly lower amounts of GFs were detected in nonactivated PRP and PPP compared to the differently activated PRP ( ).

The effects of the different activation methods on PRP GF release are shown in Figure 2 in detail.

3.3.1. PDGF

At 15 and 30 minutes, thrombin and CaCl2/thrombin produced a significantly higher amount of PDGF with respect to that of CaCl2 ( ). After 1 h, CaCl2 and thrombin alone produced a similar amount of PDGF, whereas the combination of CaCl2/thrombin induced a significantly higher PDGF release with respect to that of CaCl2 ( ). Moreover, thrombin and CaCl2/thrombin showed greater amount of PDGF with respect to that of collagen type I ( ). At 2 hours CaCl2, thrombin, and CaCl2/thrombin produced similar levels of PDGF, all significantly higher compared to those of collagen type I. After 24 h, PDGF release from CaCl2 activated PRP was significantly higher with respect to that of thrombin ( ), and CaCl2 and CaCl2/thrombin induced more PDGF compared to that of collagen type I ( ) (Figure 2).

3.3.2. TGF-β

At 15 minutes, thrombin and CaCl2/thrombin showed a greater amount of TGF-β with respect to that of CaCl2 and collagen type I ( ), whereas no significant difference was noted between CaCl2 and collagen type I. After 30 minutes, thrombin and CaCl2/thrombin produced a significantly higher amount of TGF-β with respect to collagen type I ( ). At 1 hour, thrombin induced a significantly higher TGF-β release with respect to that of CaCl2 ( ), while CaCl2, thrombin, and CaCl2/thrombin were higher with respect to that of collagen type I ( ). Significantly higher levels of TGF-β were observed for CaCl2, thrombin, and CaCl2/thrombin with respect to that of collagen type I at both 2 and 24 hours ( ) (Figure 2).

3.3.3. VEGF

At 15 and 30 minutes, thrombin and CaCl2/thrombin produced a significantly higher amount of VEGF with respect to that of CaCl2 and collagen type I ( ). After 1 hour, CaCl2 and thrombin alone produced a similar amount of VEGF, whereas the combination of CaCl2/thrombin induced a significantly higher VEGF release with respect to that of CaCl2 ( ). Moreover, CaCl2, thrombin, and CaCl2/thrombin showed a greater amount of VEGF with respect to that of collagen type I ( ). At both 2 h and 24 hours CaCl2, thrombin, and CaCl2/thrombin produced significantly higher VEGF levels compared to those of collagen type I ( ). Finally, CaCl2/thrombin produced a significantly higher amount of VEGF with respect to that of thrombin at 24 hours ( ) (Figure 2).

3.4. Growth Factor Release Kinetics

The release pattern of PRP activated with CaCl2 was similar for all the GFs evaluated, with a significant and progressive release of GFs starting from 15 minutes and increasing up to 24 hours ( ) (Figure 2). Thrombin, CaCL2/thrombin, and collagen-type-I-activated PRP showed an immediate release of PDGF and TGF- that remained stable over time (Figure 2) conversely, VEGF showed an increasing trend from 15 minutes up to 24 hours ( ).

4. Discussion

The main finding of our study is that the activation modality influences PRP clot formation, leading to differences in terms of both amount and release kinetics of platelet-derived GFs.

The most commonly used activation methods in the current clinical practice [14–16] were directly compared: CaCl2, autologous thrombin, their combination, and collagen type I to mimic the clinical conditions where their presence in the treated connective tissues should induce an “in situ” platelet activation. The latter is currently chosen for several PRP applications, since it is considered to be an easier and more effective strategy to deliver platelet bioactive molecules. However, our data showed that collagen type I does not lead to the same PRP releasate with respect to the other activators in terms of GFs released. In this study, we evaluated 3 GFs chosen among the most representative of PRP and involved in the wound healing cascade (TGF-β1, PDGF-AB, and VEGF) and 2 inflammatory mediators (IL-1β and TNF-α) to test whether the selected activators might induce an inflammatory component of PRP releasate. The in vitro results showed significantly lower quantities of TGF-β1, PDGF-AB, and VEGF when collagen type I was used in this experimental condition.

Collagen is a weak platelet activator, which results in a lower amount of GFs released with respect to the other activation methods. This is a key aspect to bear in mind, since GFs are potent molecules and even small variations might affect the results in the tissue healing process [17, 18]. In fact, although low concentrations may be not effective enough to elicit the desired effects, high GF concentrations may have inhibitory effects on cellular functions and the level of the healing response [19–21]. Besides, they can be associated with unresolved inflammation and fibrotic events [22], thus confirming the importance of obtaining a proper releasate, also by choosing a specific PRP activation method to stimulate the release of bioactive molecules according to the requirements of the targeted tissue.

Besides the overall higher amount of released GFs with the other activation strategies used in the clinical practice, the comparison of the amount of molecules detected at each time point underlined another key factor related to PRP activation: the different release kinetics. This is of major importance and may also affect the treatment outcome. In fact, a rapid activation has been associated with a decrease in the total amount of GFs available at the tissue site over time [23]. GFs have a short half-life (from minutes to hours) and, if they are not immediately used upon release from platelets, they might be degraded before additional tissue receptors become available [15, 24]. From a clinical point of view, this aspect may be one of the causes related to the poor results sometimes reported by using PRP in musculoskeletal tissue regeneration [23]. Conversely, some other applications may benefit from a less sustained release, with the final results determined by the burst of bioactive molecules released [25, 26].

The study results highlighted that thrombin alone and in combination with CaCl2 and collagen type I (even if at lower level in this case) presented similar kinetics, stimulating a rapid release of GFs that remains stable up to 24 h. Similarly, comparing the PRP releasate induced by thrombin or collagen type I, Fufa et al. [14] observed that both activation methods stimulate immediate initial release sustained over 10 days from a PRP clot.

Conversely, CaCl2 showed a gradual release over time, with a lower initial level followed by a progressively increasing amount of GFs released, reaching similar or even higher levels at the 24-hour evaluation.

Another important aspect for the clinical application of blood derivatives is their physical form, which may range from liquid to solid gel allowing both surgical augmentations as well as minimally invasive injective PRP delivery. Concerning this, the study underlined how different activators influence platelet aggregation. In particular, the use of CaCl2 induced clot formation within 30 minutes of its addition, whereas thrombin and CaCl2/thrombin caused a more rapid clot formation, which was already detectable at the 15-minute evaluation. Interestingly, collagen-type-I-activated platelet concentrates exhibited far less aggregation, with no visible clots up to 24 hours. This result is partially in contrast to that obtained by Fufa et al. [14], who observed that PRP activation with collagen led to clot formation, albeit far less retracted than that observed with thrombin activation. This discrepancy may be due to the different experimental conditions, such as the type of collagen and the different procedures used to prepare PRP. PRP may present a wide range in terms of type and quantity of cells and molecules such as fibrinogen, which may explain the different propensity to form clots. The state of the platelet concentrate may be as important as the released molecules for treatment success. In fact, although the lack of a clot might not be a problem in the treatment of osteoarthritis, where a liquid PRP allows all articular tissues to be targeted without the risk of dispersion from the closed joint cavity [5], the liquid form may be unsuitable for other applications. This appears to be clear for surgical augmentations, where PRP is used to entrap cells or even sutured to the lesion site [23], but may also apply for less invasive injective approaches. This may be the case of intratendinous injections. Once delivered inside the tendon in the liquid form, the timely gelification process may allow the concentrate to remain in the injected area, thus allowing GF secretion in the treatment site. Conversely, the persistent liquid state may increase the risk of leakage and PRP dispersion, favoured by the contraction of the musculotendinous unit that might squeeze liquid PRP away from the injection site, thus reducing or even impairing its potentially positive effects [13]. A direct PRP activation in situ is an interesting approach that might overcome some of the shortcomings related to the use of thrombin, resulting in the risk of potentially life-threatening coagulopathies [27, 28], and CaCl2, sometimes associated with burning sensation due to low pH, as reported by DeLong et al. [23]. However, the results obtained in this study with collagen-mediated activation cast doubts on this method for PRP application in the clinical practice. Further studies should focus not only on the distribution of the injected PRP in the treated area, but also on its persistence over time and on the consequent effects on the final outcome for each specific application [29].

Finally, besides the differences in clot formation and GFs amount and release kinetics, another interesting finding that emerged from the study analysis regards the lack of influence of the activation methods on the inflammatory molecules in the releasate. The PRP used in this experimental setting is a leukocyte-rich PRP, which is currently being debated for the potentially deleterious effects of proteases and reactive oxygen species released by the white blood cell component. In fact, whereas some authors consider leukocytes to be a beneficial source of cytokines and enzymes that may be important for the prevention of infection, others attribute better results to formulations with leukocyte depletion [30–32].

The study results showed that, even in this leukocyte-rich PRP, none of the selected activators was able to induce an inflammatory releasate, as demonstrated by the lack of IL-1β and TNF-α secretion at all the experimental times evaluated. Future studies should investigate if the same findings will be confirmed also in an inflammatory environment better reproducing PRP use in the damaged tissues of the clinical setting.

This study has some limitations that need to be discussed. In fact, today little is known about the concentration of calcium, thrombin, or collagen needed to trigger the optimal release of GFs, and different concentrations may lead to different results. For example, it has been reported that high concentrations of calcium and thrombin trigger an immediate and significant increase in TGF-β1 and PDGF concentrations, which remained generally constant over a 6-day period, whereas lower concentrations tend to reduce and delay GF release [26]. However, the selected concentrations derive from the clinical practice thus, the study findings still reflect the current PRP applications. Nonetheless, the activator concentration should be the focus of further specifically designed studies, being a key aspect to determine the final properties of the platelet concentrates. Moreover, these findings give only general indications that could be useful for future PRP application in musculoskeletal tissues, and further studies are needed to investigate the effects of different PRP activation methods on cell cultures.

The results of this study confirm the importance of the method chosen to activate PRP, by determining both its physical form and the amount and release kinetics of GFs. It is not only the presence of GFs that dictates the level of healing response, but also the ability of targeting the treatment area, thus modulating cells with an appropriate dosage and in a timely manner [33]. Thus, PRP activation strategies should be selected not only based on procedure type (open versus arthroscopic), but also according to the desired biological effects in the targeted tissue. Future studies should aim at further investigating the effect of the different activation strategy on platelet concentrates according to the lesion target, in order to optimize the in vivo effect of the released bioactive molecules and therefore increase PRP healing potential.

Competing Interests

Giuseppe Filardo is consultant and receives institutional support from Finceramica Faenza SpA (Italy), Fidia Farmaceutici SpA (Italy), and CartiHeal (2009) Ltd. (Israel). He is a consultant for EON Medica SRL (Italy). He receives institutional support from IGEA Clinical Biophysics (Italy), BIOMET (USA), and Kensey Nash (USA). Elizaveta Kon is a consultant for CartiHeal (2009) Ltd. (Israel) and has stocks of CartiHeal (2009) Ltd. (Israel). She is a consultant and receives institutional support from Finceramica Faenza SpA (Italy). She receives institutional support from Fidia Farmaceutici SpA (Italy), IGEA Clinical Biophysics (Italy), BIOMET (USA), and Kensey Nash (USA). Maurilio Marcacci receives royalties and research institutional support from Fin-Ceramica Faenza SpA (Italy). He receives institutional support from Fidia Farmaceutici SpA (Italy), CartiHeal (2009) Ltd. (Israel), IGEA Clinical Biophysics (Italy), BIOMET (USA), and Kensey Nash (USA). All the other authors declare that there are no competing interests regarding the publication of this paper.

Acknowledgments

The authors thank Dr. Elettra Pignotti for statistical assistance and Mr. Keith Smith for English editing, Rizzoli Orthopaedic Institute, Bologna, Italy. This work was supported by grants from “

” funds, Italian Ministry of Health (Project RF-2009, Grant no. 1498841), and Emilia-Romagna Region (Region-University Program 2010–2012: Regenerative Medicine of Cartilage and Bone).

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Copyright

Copyright © 2016 Carola Cavallo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


1. Avoid Transferring DC During Culture

Dendritic cells are composed of both adherent and non-adherent cells, making the movement of cells from one culture vessel to another a dicey situation. Those adherent cells won’t come off the plastic without a real fight. When possible, set up your cells in the culture configuration you want to use in your experiment, so you don’t have to disturb the cells.
If you do switch vessels during your culture or experimentation, expect to recover fewer cells.

2. Add the Right Proportion of Cytokines to Preserve the DC Phenotype

Dendritic cells will survive in culture for approximately one week. If you have plans to maintain your dendritic cells after an overnight culture, you need to add the proper proportion of granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL4) recombinant human proteins.

We recommend adding 500 U/mL of GM-CSF and 500 U/mL of IL4, which you can purchase from suppliers like Thermo Fisher or PeproTech. This will ensure your dendritic cells maintain their phenotype after thawing.

3. Activate Your DC, If Necessary

Our dendritic cells are immature, but you can easily activate or mature them by adding lipopolysaccharides (LPS), poly(I:C), or other pathogen pattern molecules. Alternatively, you could use cytokines, such as IFNγ or TNFα, in combination with prostaglandin to achieve a similar result.

Depending on the stimuli used, you can get a slightly different dendritic cell that could suppress an immune response rather than ramp it up.

4. Always Include Serum When Stimulating DC with LPS

You can stimulate your dendritic cells with LPS, but you must include serum as well for proper activation. The soluble CD14 in the serum facilitates binding of the LPS to the dendritic cells.

For serum, you can use human serum or fetal bovine serum to achieve the desired result.

5. Follow Our Detailed Protocol for Successful DC Cultures

In addition to these tips, be sure to follow the procedures outlined in our Human Dendritic Cell Culture protocol.
Have other research questions? Ask a scientist and get a quick answer!

Author: Anne Lodge, Ph.D.

Dr. Lodge is the Chief Science and Innovation Officer at Cellero. She has a strong background in cell-based therapeutics and immunology, including a Ph.D. in Cell and Molecular Biology from the University of Vermont and a postdoctoral fellowship with the Multiple Sclerosis Society studying the role of T cells in the disease process. Learn more about Dr. Lodge.

2 Comments

Hi, thank you for these tips. I want to ask something. As far a I know we couldn’t make cryopreserved storage of dendritic cells. Than how long can we culture these cells by changing the media?

If you are starting from monocytes, it takes 4-6 days before they fully differentiate into DC (when culturing with GM-CSF and IL-4). This is the best time to harvest the DCs for your experiment. You can add fresh media to maintain them in culture for 2-3 more days. However, these cells will be increasingly adherent to tissue culture plastic.

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Platelets in atherothrombosis

The participation of platelets in atherogenesis and the subsequent formation of occlusive thrombi depend on platelets' adhesive properties and the inability to respond to stimuli with rapid activation. By understanding the multifaceted mechanisms involved in platelet interactions with vascular surfaces and aggregation, new approaches can be tailored to selectively inhibit the pathways most relevant to the pathological aspects of atherothrombosis.

Arterial thrombosis is the acute complication that develops on the chronic lesions of atherosclerosis and causes heart attack and stroke, today the most common causes of mortality in developed countries. Platelets, with fibrin, are prominent components of the thrombi (clots) that occlude arteries, but may also participate in the development and progression of the atherosclerotic plaque. Thus, platelets are central to the process of atherothrombosis, a term that describes the combination of acute and chronic events in arterial disease. The normal function of platelets, however, is to arrest bleeding from wounds, which requires adhesion to altered vascular surfaces and rapid cellular activation with the ensuing accumulation of additional platelets and fibrin into a growing thrombus. Progress in understanding the mechanisms of platelet adhesion, activation and aggregation may improve the ability to prevent thrombosis and, possibly, the progression of atherosclerosis.