Supplemental Digital Content is available in the text Currently, the combination of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) is the standard therapy for metastatic pancreatic cancer. In recent years, FOLFIRINOX-based neoadjuvant therapy for locally advanced pancreatic cancer (LAPC) has been gaining an increasing amount of attention, owing to its ability to reduce disease stage and transform LAPC to borderline resectable or even resectable pancreatic cancer. Accordingly, we aimed to evaluate the efficacy of first-line FOLFIRINOX chemotherapy in patients with LAPC.We searched PubMed, Embase, and Cochrane Library from the time of establishment till January 1, 2020 and included studies focusing on LAPC patients who received FOLFIRINOX as first-line neoadjuvant treatment. The primary outcomes were: resection rate and radical (R0) resection rate while the secondary outcomes were: objective response rate, overall survival, progression-free survival, and rate of grade 3 to 4 adverse events. The meta package for R 3.6.2 was used for heterogeneity and publication bias testing.Twenty-one studies, including 653 patients with LAPC, were selected. After treatment with FOLFIRINOX, the resection rate was 26%  and R0 resection rate was 88% . The response rate was 34% . The median overall survival and progression-free survival durations ranged from 10.0 to 32.7 months and 3.0 to 25.3 months, respectively. The observed grade 3 to 4 adverse events were neutropenia , febrile neutropenia , thrombocytopenia , nausea/vomiting , diarrhea , and fatigue .FOLFIRINOX-based neoadjuvant chemotherapy has the potential to improve the rates of resection, R0 resection, and median OS in LAPC. Our results require further validation in large, high-quality randomized controlled trials. The diagnosis of this disease in the early stages is difficult and only 15% to 20% of patients undergo surgery at first diagnosis.,5 However, even after radical surgery, the 5-year survival rate associated with the disease is only 15% to 25%.Pancreatic cancer (PC) is among the most malignant cancers, characterized by rapid progression, poor prognoses, high postoperative recurrence rates, and 5-year survival rates lower than 5%.–9 The combination of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) is increasingly gaining importance in the treatment of metastatic pancreatic cancer (MPC). A randomized controlled trial (RCT) from the United States (PROD-IGY 4/ACCORD 11) showed that MPC patients treated with FOLFIRINOX had a better objective response rate (ORR)  and median survival duration  than those with gemcitabine monotherapy. In addition, FOLFIRINOX can improve the quality of life of MPC patients compared with gemcitabine alone. Given its efficacy in MPC treatment, FOLFIRINOX is now widely used for patients with LAPC, as it has the potential to reduce the disease stage, transform it to borderline resectable or even resectable pancreatic cancer, and improve the associated surgical resection rate. Furthermore, a meta-analysis also indicated that FOLFIRINOX had an advantage in improving the median overall survival (OS) of patients with LAPC. However, most previous studies on the topic had an insufficient sample size, owing to which definitive conclusions about the efficacy and safety of FOLFIRINOX in LAPC patients could not be drawn.Neoadjuvant chemotherapy for PC, a hot topic in clinical research, has been shown to improve the prognoses of patients, through reductions in the development rate of tumor lesions, increases in the R0 resection rate, reductions in the rates of vascular invasion and micrometastasis, and decreases in the incidence of postoperative complications.The aim of the present meta-analysis was to assess the efficacy of FOLFIRINOX as a first-line chemotherapy regimen for patients with LAPC.22.1This meta-analysis was PRISMA-compliant. The ethics committee  approved the study. We systematically searched PubMed, EMBASE, and the Cochrane Library for relevant studies published from database establishment to January 1, 2020, including RCTs, clinical controlled studies, and cohort studies, among others. The search terms included “FOLFIRINOX,” “fluorouracil,” “irinotecan,” “oxaliplatin,” “pancreatic cancer,” “pancreatic neoplasm,” “drug combination,” and relevant variants thereof. Only studies published in English were included. There were no restrictions on the population, race, or publication date. Potential eligible studies were identified by searching the references of the selected studies. The search was completed independently by 3 authors. Specific details on the search strategy are presented in Appendix 1, Supplemental Content.2.2All the included studies focused on the effectiveness of FOLFIRINOX in patients with LAPC. The following selection criteria were applied for the included studies: study design: RCT, cohort study, clinical controlled study, etc, presence of at least 1 patient with LAPC, accurate LAPC diagnosis by imaging or pathology prior to chemotherapy, and use of FOLFIRINOX as the first-line chemotherapy regimen for LAPC. In addition, studies were excluded if they met the following criteria: study design: case report, review, conference abstract, and republication, lack of data on resection rate or R0 resection rate, and insufficient data on the outcome of interest, or impossibility of the calculation of the outcome of interest. Two reviewers assessed the titles and abstracts independently for eligibility, and the full tests were further assessed to check if they met the selection criteria. Disagreements were resolved through discussions with a third reviewer. A predefined data collection form was used for the extraction of data from the selected studies. The primary outcomes were the rates of resection and R0 resection after first-line FOLFIRINOX treatment for LAPC. The secondary outcomes were the rates of response, median OS, median progression-free survival (PFS), and grade 3 to 4 adverse events. Other collected information included that on the first author, year of publication, type of study, total sample size, number of patients treated with FOLFIRINOX, median age, performance status, treatment regimen, tumor stage, median chemotherapy duration, and follow-up duration. The characteristics of the selected studies are shown in Table 2.3 The NOS comprises 3 factors: patient selection, comparability of the study groups, and evaluation of results, including 8 items with a full score of 9. A score lower than 4 is indicative of a low study quality and that higher than 7 reflects a high quality. The NOS scores of the included studies are shown in Table The methodological quality of the included studies was evaluated independently according to the Newcastle–Ottawa scale (NOS) by 2 reviewers.2.4χ2-based Q test and I2 test.,16 The level of heterogeneity was considered to be significantly different at I2 > 50% or P < .1 in the Q test. According to Cochrane review guidelines, the level of heterogeneity was considered significant at I2 > 50%, and the random effects model was selected. Otherwise, the fixed effects model was used to evaluate the 95% confidence intervals (CIs). Additionally, sensitivity analyses of the surgical resection rates were performed by the removal of each study for the assessment of the quality and stability of the results. P < .1 was considered statistically significant. Publication bias was assessed using funnel plots  criteria in 3 studies, and other criteria in 9 studies. Sixteen studies reported the median number of FOLFIRINOX cycles administered to patients with LAPC, which ranged from 4.9 to 11.5. And of these studies, 12 reported the median number of FOLFIRINOX cycles was 6 or higher. In the 12 studies, 280 patients received additional radiation after chemotherapy, and in 6 of these, 146 patients received it in combination with gemcitabine, oxaliplatin, capecitabine, or 5-fluorouracil as a radiosensitize, Thirty-one patients from 2 studies were administered stereotactic radiotherapy, of whom 12 patients received a dose of 36 Gy in 3 fractions and the other 19 received a dose of 33 Gy in 5 fractions. Furthermore, 6 patients in 1 study received intraoperative radiotherapy.The characteristics of the included studies are shown in Table 3.2P = .12). Meanwhile, the R0 resection rate in 10 studies, in which the patients received 6 or fewer median FOLFIRINOX cycles was 90%  compared with the 88%  observed in 3 studies in which the median number of cycles was lower than 6 (P = .81). All the studies used a random-effects model and the forest plots are shown in Figures A total of 648 LAPC patients from 21 studies were analyzed. In total, 190 (29%) LAPC patients underwent surgical resection after first-line FOLFIRINOX chemotherapy ± radiotherapy, and the pooled resection rate was 26% , with a random-effects model . Only 1 study reported a toxic death attributed to FOLFIRINOX and the most likely cause of death was pulmonary embolism. The most commonly reported grade 3 to 4 adverse hematological events included neutropenia, febrile neutropenia, and thrombocytopenia Table ; the cor4 In addition, previous studies have shown that objective efficiency can improve patients’ prognoses and survival, suggesting that the FOLFIRINOX approach rather than the gemcitabine-based approach may have potential benefits, in terms of survival outcomes.,41In our meta-analysis, which included 21 studies involving 648 patients with LAPC who received FOLFIRINOX as first-line chemotherapy, we observed an ORR of 34% , which was significantly higher than the value associated with gemcitabine treatment (9.4%).,43; therefore, palliative chemotherapy has become the mainstay in the treatment of advanced PC. In 1997, a randomized trial by Burris et al confirmed that patients receiving gemcitabine monotherapy had a slight advantage in terms of median OS over those receiving 5-fluorouracil monotherapy in LAPC and MPC settings . In the years that followed, gemcitabine became the standard treatment for MPC and LAPC. Chauffert et al evaluated gemcitabine as a first-line treatment for LAPC, and showed a median OS duration of 6 to 13 months. However, in 2011, an RCT showed that FOLFIRINOX treatment improved the PFS (6.4 vs 3.3 months) and OS (11.1 vs 6.8 months) durations in MPC patients to a greater degree than gemcitabine monotherapy. As a majority of LAPC patients show better performance rates than MPC patients, a growing number of studies are now using FOLFIRINOX as the first-line chemotherapy regimen for patients with LAPC. Suker et al conducted a patient-level meta-analysis that evaluated the role of FOLFIRINOX in LAPC patients, including 11 studies involving 315 patients, and demonstrated a median OS duration of 24.0 months (95% CI = 21.7–26.8 months). Subsequently, Suker et al conducted a cohort study that contrasted with the gemcitabine scheme investigated by Chauffert et al, and showed that FOLFIRINOX treatment in LAPC patients resulted in longer median OS durations; their work significantly contributed to the use of FOLFIRINOX in such settings. It follows that LAPC patients have longer survival durations than MPC patients after treatment with FOLFIRINOX. Compared with the values observed by Chauffert et al, our meta-analysis of 21 studies reported median OS values ranging from 10.0 to 32.7 months and median PFS values ranging from 3.0 to 25.3 months in LAPC patients. Our results indicate that FOLFIRINOX exhibits stronger efficacy than gemcitabine in LAPC patients.PC is systemic in nature and up to 85% of those with the disease are diagnosed with tumors that involve local arteries or distant metastases Therefore, for a large number of patients with LAPC, surgical resection yields the highest long-term values. In the past, only 1% to 5% of LAPC patients underwent complete surgical resection after single-drug neoadjuvant chemotherapy.,49 Fortunately, a growing number of studies are now reporting that LAPC patients who receive neoadjuvant FOLFIRINOX may potentially be able to undergo surgical resection as a result of tumor downstaging. All the 21 studies included in the current meta-analysis reported surgical resection rates ranging from 6.3% to 61.1% in LAPC patients with neoadjuvant FOLFIRINOX chemotherapy. The surgical resection rates reported across different studies show an obvious degree of heterogeneity. At the same time, in our sensitivity analysis of the rate of surgical resection, we found that when 2 studies were excluded , the level of heterogeneity of the results decreased, but there was no directional change. One reason for this difference may be the lack of consensus regarding the resectability criteria after neoadjuvant therapy. Therefore, for the performance of more accurate comparisons, future studies may need to reach a consensus on the resectability criteria. In the present meta-analysis, the surgical resection rate in the LAPC patients with FOLFIRINOX as first-line chemotherapy was 26% and 88% of these patients underwent R0 resection. The R0 resection rate was even higher than that reported by Gillen et al in patients with a resectable status (88% vs 80%). This indicates that the use of the FOLFIRINOX regimen significantly increases the chance of R0 resection in LAPC patients. In addition, we performed subgroup analyses of the surgical resection rates and R0 resection rates using the median number of FOLFIRINOX cycles as a grouping factor to evaluate whether the number of FOLFIRINOX cycles influences the rates of surgical resection and R0 resection; however, no statistically significant differences were observed. This may be attributed to the small sample size of the included studies. In addition, Janssen et al showed that a median number of FOLFIRINOX cycles were greater than or equal to 6, with a median OS of 21.4 months (95% CI = 16.7–36.0 months), compared with those observed of 21.7 months (95% CI = 15.0–28.4 months) in a study with a median number of chemotherapy cycles lower than 6, with no statistical difference. Therefore, further studies are required to investigate whether the median number of FOLFIRINOX cycles is an important factor in the determination of the rates of surgical resection and median OS.PC is associated with high mortality values, and most patients with early-stage disease tend to die. Surgical resection is the only chance for cure in PC, although few patients are eligible for surgery. In a previous meta-analysis of PC patients who underwent surgical resection, a survival duration of 3 to 5 years was observed, which was longer than that noted among those who did not undergo surgical resection. Meanwhile, in another recently published study, 26% of 215 patients with LAPC who received chemoradiotherapy underwent surgical resection and 10% achieved R0 resection. Thus, although the use of radiotherapy or chemoradiotherapy may be convincing for local control, the unresectable status persists after treatment in many patients. Therefore, the role of these treatments in locally advanced disease needs further clarification in future studies.In addition, in terms of the rate of local tumor progression, we found that 28% of the patients received additional radiotherapy or chemoradiotherapy after FOLFIRINOX treatment. There was no significant difference in the degree of resectability between patients who received radiotherapy or chemoradiotherapy and those who did not. A meta-analysis of 14 phase II clinical trials showed that the rates of surgical resection and R0 resection were 32% and 20%, respectively, in LAPC patients after neoadjuvant chemotherapy, with or without radiotherapy.Although the effect of FOLFIRINOX is superior to that of gemcitabine in patients with metastatic PC, its toxicity somewhat hinders its clinical application. In our study, the most commonly observed grade 3 to 4 adverse event was neutropenia, which showed an incidence that was similar to that reported by Suker et al (20% vs 19.6%). Granulocyte-colony stimulating factor is widely used in the prevention of this hematological toxicity. In addition, in a majority of the studies we included, the dose of the chemotherapeutic drugs was reduced based on the patients’ tolerance level, with the aim of reducing the rates of adverse events and improving the efficacy of chemotherapy. It has been demonstrated that the modified FOLFIRINOX regimen yields satisfactory results in patients with an ECOG performance status score of 0 to 1.This study has some limitations that should be considered. First, the sample size of the included studies was relatively small, with a high level of heterogeneity, and a majority of the studies had a retrospective design , which may challenge the accuracy of the study results and lead to their overestimation. Second, different criteria were used in the diagnosis of LAPC. Most of the studies referred to NCCN or AHPBA/SSO/SSAT criteria, while some used other criteria, affecting the determination of the outcome indicators. Third, a majority of the studies did not clearly report the implementation details of the FOLFIRINOX-based treatment regimens and involved reductions in the dose of the chemotherapeutic drugs. The presence of heterogeneity across the studies may have biased the results. Fourth, the included studies did not report on the ethnicities and dietary habits of the study populations, owing to which the comprehensiveness of the results is low.In conclusion, FOLFIRINOX-based neoadjuvant chemotherapy can improve the rates of resection, R0 resection, and median OS in LAPC. These results require further validation in large, high-quality RCTs.Conceptualization: Tianwu Yu.Data curation: Zhiliang Chen, Rui Diao.Formal analysis: Zhiliang, Chen, Yongshuang, Lv.Methodology: Yongshuang Lv, Jian, Zhou.Software: Zhiliang, Chen.Supervision: Tianwu, Yu.Writing – original draft: Zhiliang, Chen, Yongshuang, Lv.Writing – review & editing: He Li, Tianwu Yu.


To investigate the effect of Shenfu (SF) injection on donor heart preservation.Twelve pigs were randomly divided into SF group (n=6) and control group (n=6). After eight hours of perfusion, the differences in hemoglobin, the expression of Bcl-2 and BAX, and changes in the myocardial ultrastructure were compared to illustrate the effects of SF injection in heart preservation.P=0.001), and there was significant interaction of groups with times (P=0.019), but the perfusion time may not be associated with the hemoglobin concentration (P=0.616). According to Western blotting analysis, the expression of Bcl-2 was higher in the SF group than in the control group, while the expression of BAX was not different between the two groups. As to ultrastructural changes, both groups exhibited mitochondrial swelling and myofilament lysis, but the degree of damage in the SF group was smaller.The differences in free hemoglobin between the SF group and the control group were statistically significant (Our study suggests that the application of SF injection for heart preservation may protect against cardiomyocytes and erythrocytes apoptosis, and Bcl-2 protein may play a role in these physiological processes. In cold static storage, ischemia-reperfusion injury is inevitable, and continuous perfusion of harvested hearts can provide a near-physiologic environment for the heart by continuously infusing it with a warm, oxygen-rich medium. It cannot only provide sufficient nutrients, but also ensure the effective removal of metabolic waste. Furthermore, it also can lighten the ischemia-reperfusion injury induced by low temperature and avoid hyperkalemia. However, challenges still exist. Immune cells are infused with erythrocytes, metabolic substrates, and other beneficial substances, thus promoting a pro-inflammatory cytokine milieu after exposure to the ex vivo circuit,5. The inflammatory environment has an adverse effect on the myocardium, vascular intima, and erythrocytes. Furthermore, severe mechanical damage and inflammatory mediators attack erythrocytes, resulting in increased free hemoglobin in the blood, which causes free hydroxyl production and lipid peroxidation, then leading to tissue damage.In order to optimize the effect of heart preservation, new approaches have been explored. For instance, continuous perfusion of harvested hearts can safely prolong the preservation period compared with cold static storage,7. With the function of increasing blood flow, Shenfu (SF) injection may enhance the tolerance of organs to hypoxia. Previous studies suggested that SF injection can improve myocardial metabolism by mediating the expression of Bcl-2 protein and inhibit myocardial ischemia-reperfusion injury in diabetic rats. In addition, SF injection is also identified to be involved in the immune dysfunction by modulating the expression of complements and cytokines levels, and Chen et al. report that SF injection may play a protective role in ischemia-reperfusion injury of liver transplantation. SF injection mainly consists of Panax ginseng and Aconitum carmichaeli. Panax ginseng is the steamed root of Panax ginseng C. A. Meyer and belongs to the family Araliaceae with the characteristics of a medicinal preparation in some countries. The active ingredients of Panax ginseng are the ginsenosides with a dammarane skeleton, which belong to dammarane type and include ginsenosides Rb1, Rb2, Rc, Rd, Rg1, Re, Rf, Rg2, and Rg3. Recently, ginsenosides are reported to protect myocardial ultrastructure and reduce Ca2+ overload. Aconitum carmichaeli is associated with heart rate, rhythm, blood pressure, and hemodynamics, and can protect myocardial cells. Higenamine is the potent alkaloid isolated from Aconitum carmichaeli, with inotropic and chronotropic effects in cardiovascular system. Taken together, we hypothesized that the use of SF injection in continuous perfusion would attenuate the inflammatory response, improve microcirculation, and protect against myocardial injury, and this study was conducted to confirm this hypothesis.Some traditional Chinese medicines are used as additives for heart preservation and can alleviate myocardial ischemia-reperfusion injury to some extent. Twelve Guangxi Bama miniature pigs weighing 25-30 Kg were randomly divided into experimental group and control group.Our Institutional Animal Care approved this study, and all animals were disposed according to the “Guide for the Care and Use of Laboratory Animals”After the animals were anesthetized by intramuscular injection of diazepam (1 mg/Kg) and ketamine (30 mg/Kg), a 24G venous indwelling needle was used to establish venous access via marginal ear vein. All pigs were intubated and mechanically ventilated. The respiratory rate was controlled at 20 times per minute, the tidal volume was 10 ml/Kg, and the inspiratory/expiratory ratio was 1:2. 1% to 2% isoflurane were used to maintain the anesthesia state. To perform hemodynamic monitoring and support, the right carotid artery and vein were cannulated. Then a median sternotomy was conducted. A blood crossmatch procedure was performed between the donor and the recipient to confirm the accurate matching prior to transfusion in case of insufficient blood supply.After the great vessels were separated from the pericardium, heparin (400 U/Kg) was infused intravenously. The intact pericardium was preserved. Next, the brachiocephalic artery was cut off and the perfusion tube was inserted. Then, the donor blood was harvested through the superior vena cava and the right superior pulmonary vein, which was filtrated through leukocyte-depleting filters. After being arrested by 30 mL/Kg University of Wisconsin solution , the initial part of descending aorta and the two branches of aortic arch were cut off, and then the heart was removed. The drainage tube was placed through the pulmonary vein and the mitral valve, and the heart was inverted to achieve better decompression., and mixed with 5 mL/Kg hydroxyethyl starch  to perform oxygenation and temperature change. The perfusate consisted of sodium potassium magnesium calcium and glucose injection  and leukocyte-depleted blood (600 to 800 mL) with a total of 1100 to 1300 mL, and hematocrit was controlled at 20 to 25%. The SF injections  were additionally applied in the experimental group by pre-filling in membrane oxygenation (0.4 mL/Kg/h) and continuous infusion (0.2 mL/Kg/h). The blood in the perfusion circuit was oxygenated with 95% oxygen and 5% carbon dioxide with a hollow-fiber membrane oxygenator . Blood perfusion began within 10 minutes after arrest and was maintained at 32 ºC to 35 ºC for eight hours. The hemoglobin concentration in perfusate was detected at T0, T1 (one hour after blood perfusion), T2 (two hours after blood perfusion), T3 (three hours after blood perfusion), T4 (four hours after blood perfusion), T5 (five hours after blood perfusion), T6 (six hours after blood perfusion), T7 (seven hours after blood perfusion), and T8 (eight hours after blood perfusion) during preservation with hemoglobin colorimetric assay kit . The perfusion flow was adjusted to achieve 60-80 mL/min and the perfusion pressure was controlled at 40-60 mmHg.After filtrated out the leukocyte, the harvested blood was put into the perfusion device, which was described as a small cardiopulmonary bypassAfter continuous infusion for eight hours, a small piece of the left ventricular myocardium was homogenized in a cold lysis buffer supplemented with protease inhibitors, and protein concentrations were evaluated using a bicinchoninic acid protein assay kit  with Tubulin as loading control. The samples of tissue lysis were heated to 95 °C for five minutes. Protein samples (40 µg) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted onto polyvinylidene fluoride membranes overnight at 4 °C. Membranes were incubated with the appropriate primary antibody including anti-Bcl-2, anti-BAX, and anti-Tubulin, all from Elabscience, overnight at 4 °C. After washing with tris-buffered saline (TBST) three times, five minutes for each time, the secondary anti-rabbit immunoglobulin G antibody was then added and incubated for 30 minutes. After washing with TBST three times, proper amount of electrochemical luminescent substrate was added and incubated with the membranes in the dark. The results were scanned and processed by the ImageJ software.,18, of which glutaraldehyde and osmium tetroxide were applied as fixatives and spur was used as resin. A transmission electron microscope was applied for the ultrastructural findings of biopsies.After preservation for eight hours and cardioplegic arrest , left ventricular free walls were taken from two donor hearts from the experimental group and the control group. The tissues were processed into blocks for transmission electron microscopyP-value of < 0.05 was identified as statistical significance. The IBM SPSS Statistics 22.0 software was used to perform these analyses.Measurement data were described as mean ± standard deviation, and differences among time points in the group were compared using one-way repeated measures analysis of variance (ANOVA). All statistical analyses were two-tailed, and a P>0.05). The total perfusate, anastomotic time, perfusion pressure, and perfusion flow were also not different (P>0.05).Basic characteristics of the experimental animals are shown in P<0.001). So, the results of multivariate tests were adopted. According to these results, the differences in free hemoglobin between the SF group and the control group were statistically significant (P=0.001), and there was significant interaction of groups with times (P=0.019), but the perfusion time may not be associated with the hemoglobin concentration (P=0.616). The results are shown in In repeated measures ANOVA, Mauchly test of sphericity failed  and can inhibit the overexpression of TNF-α messenger ribonucleic acid (mRNA) and interleukin-1 mRNA. Secondly, SF injection has been reported to play a protective role in blood vessel spasm and microcirculation. Additionally, the components of SF, ginsenosides and aconitine, exert significantly protective effect on cardiovascular system by activating α and β receptors, thereby enhancing cardiac function as well as elevating and stabilizing blood pressure to relieve reperfusion injury. However, inflammatory markers and related measurements were not investigated, and this was the limitation in our study; further studies on the abovementioned process should be focused on these. Based on the current observations and previous studies, SF injection may be used as an additive for heart preservation, which is regard as one of the commonly used drugs for the assistant treatment of cardiovascular diseases.During the perfusion process, anomalous mechanical and environmental factors influence the blood perfusion and cause damage to red blood cells (RBCs) through immediate and delayed hemolysis or the mechanical property changes of RBCs, which may impair microcirculation and tissue oxygen supply, and these processes would lead to the increase of hemoglobin concentration due to the release of RBCs,26, which is an important cellular process in myocardial ischemia-reperfusion injury. In vitro experiment demonstrated that SF injection has a protective effect on cardiac myocytes by mediating the expression of Bcl-2 protein, especially in apoptosis induced by hypoxia/reoxygenation injury. As mentioned above, ischemia-reperfusion injury is inevitable in heart preservation, so SF may exert a protective effect against this injury in perfusion process. Furthermore, the addition of anti-Bcl-2 antibodies to the RBCs that had been loaded with Bcl-2 and Bcl-2/Δ34 can induce hemoglobin release from the RBCs under proper conditions, and this phenomenon suggested that a shortened fragment of Bcl-2, Bcl-2/Δ34, may have a functional activity. BAX can promote the activation of apoptosis via multiple caspase-dependent pathways, and the collective effect of BAX and Bcl-2 would determine the initiation of mitochondrial apoptosis pathway. In our study, the expression of Bcl-2 was significantly increased in the SF group compared with the control group, and combined with the changes in concentration of hemoglobin during perfusion process, the interaction of SF injection with Bcl-2, not BAX, may play a role in cardiomyocytes and erythrocytes apoptosis.Bcl-2 family proteins participate in apoptosis in cardiomyocytes. According to the changes in the myocardial ultrastructure, the SF group suggested milder damage than the control group. A previous study reported that the compound Chinese medicine “Kang Fu Ling” can protect against the myocardial injury by inhibiting the mitochondrial permeability transition pore (MPTP) opening that induced the early ischemia-reperfusion injury secondary to calcium overload, and the inhibitions of MPTP opening and calcium overloading are the critical processes in the postischemic treatment. However, we can only identify that SF injection would have a protective effect against mitochondrial swelling in terms of the current findings, but the exact mechanisms are still uncertain, perhaps MPTP multiprotein complex is also involved in the protection of SF injection that influences the mitochondrial signal transduction.Mitochondria are extremely important organelles, which accounts for 40-60% of the myocyte volume and can be associated with cardioprotection. In our study, the perfusion flow and pressure were indistinguishable between the two groups and were controlled at 40 to 60 mmHg and 60 to 80 mL/min, respectively. These data were determined previously, and also were determined that the change of pressure from 100 to 50 mmHg would not influence oxygen uptake, coronary flow and cause ischemia, and that a perfusion pressure of 40 to 60 mmHg can improve myocardial bleeding and edema. Furthermore, lower perfusion flow would lead to hypoperfusion and deteriorate cardiac function. Therefore, our study controlled these factors to illustrate the effect of SF injection independently.Perfusion management is a critical process in the normothermic beating preservation, of which the perfusion solution, perfusion flow, and perfusion pressure are the key elementsSeveral limitations also existed in our study. Firstly, the sample size of each group was limited, and the observations should be confirmed in other experimental studies. Another limitation was that how the SF injection protects against mitochondrial swelling should be investigated in further studies.Our study suggests that the application of SF injection for heart preservation may protect against cardiomyocytes and erythrocytes apoptosis, and Bcl-2 protein may play a role in these physiological processes.


The protocol consists of 2 intramuscular injections of 0.9 mg of rhTSH, performed on day 1 day and day 2, with blood collection on day 1 (before rhTSH administration), and day 5. TSH, free thyroxine, total cholesterol, low-density lipoprotein cholesterol (LDLc), high density lipoprotein cholesterol (HDLc), and triglycerides were assessed in all the samples, before and four days after the first administration of rhTSH. Results: Total cholesterol and triglycerides significantly increased after stimulation of rhTSH . LDLc and HDLc showed comparable concentrations before and after the test , while non-HDLc increased after stimulation . Conclusion: TSH has a direct effect on total cholesterol, triglycerides, and nonHDLc. Explanation of these phenomena will require additional studies.Background: Subclinical hypothyroidism is associated with increased blood lipid levels. However, the exact role of thyrotropin (TSH) alone is not clear. In order to clarify this point, we analysed the acute effect of recombinant human TSH (rhTSH) administration on lipid levels. Methods: Sera of 27 premenopausal women with well-differentiated thyroid cancer were analysed. Patients that underwent a total thyroidectomy, ablation with  Thyroid hormones regulate different processes in the human body and the effects of their action are more evident during dysfunction of the thyroid gland, affecting processes from the cardiovascular system to cognition level ,2,3. DisWhile the serious effects of overt hypothyroidism (increased TSH and low free hormones) are well acknowledged, different studies were focused on the association between subclinical hypothyroidism and increased serum lipid concentration. Some of them found an inverse association with FT4, while others a positive and direct effect of TSH  . The issThe hypothesis that TSH can directly increase the lipids level is relatively recent, and based on associated studies not only in patients with increased TSH level , but also in subjects with TSH within the normal reference range . Indeed,131I therapy, have constant FT4 level due to their LT4 supply, which is equal every day. Recent evidence also suggests an association between TSH and hypercholesterolemia in female patients with differentiated thyroid cancer receiving LT4 after total thyroidectomy .Given the origin of the sera employed in the study , identified only by a letter  and a number (age), resulted an absolute inability to identify the subject in order to obtain the consent.t-test was used to assess the differences in each variable before and after rhTSH stimulation. Statistical significance was set at p values less than 0.05 in Stata 12.A paired p < 0.001), while FT4 values were comparable . Total cholesterol and triglycerides significantly increased after stimulation of rhTSH . LDLc showed an increase of borderline statistical significance , while HDLc showed comparable concentration before and after the test , while non-HDLc increased after stimulation .Sera of 27 females (mean age 39.4 ± 8 years) were employed in this study. Descriptive characteristics of the results, before and after rhTSH, are described in p < 0.001 for both). After rhTSH, women aged 39.4 or older still have increased total cholesterol and LDLc (p < 0.01 for both). HDL and triglycerides were comparable indecently from mean age groups, before and after rhTSH.To test lipids difference across age, we split the sample accordingly to mean age, as reported in In this study, we found that administration of rhTSH acutely increases lipids levels. In particular, we observed an increase of total cholesterol, triglycerides, and non-HDLc, while LDLc and HDLc showed no significant variations.The effect of thyroid hormone on lipid metabolism is complex and poorly understood. Lipid metabolism is regulated by different enzymes, which are regulated by different specific gene and hormones ,21 and lThyroid hormone induces the transcription of LDL receptor gene in the liver and the expression of hydroxymethylglutaryl coenzyme A reductase (HMGCR), therefore increasing cholesterol synthesis. This means that in case of hypothyroidism, the hepatic cholesterol synthesis is reduced. At the same time, triiodothyronine increases the expression of the sterol regulatory element-binding protein-2 (SREBP-2), which increases the expression of LDL receptor. Lack of thyroid hormone cause a reduction of SREBP-2 and, consequently, a reduction of LDL receptor, causing hypercholesterolemia. Hypothyroidism causes also a reduction of cholesterol ester transfer protein (CETP), which may be related to alteration in HDLc levels . The impThe lipid-lowering effect of thyroid hormone has been documented also by its use as therapeutic agent, the so-called thyromimetics. Eprotirome, one of the most studied thyromimetic drug, stimulates triiodothyronine (T3) receptor-β isoform, and results in a decreases triglycerides, LDLc, apoB, and lipoprotein lipase a .Recent evidence suggests that TSH was associated with lipids, independently of FT4. Gong et al. demonstrated that patients with subclinical hypothyroidism have increased serum PCSK9 levels, whose expression is enhanced in rhTSH-treated HepG2 cells through the activation of SREBP1c and SREBP2. Consequently, LDL uptake is decreased resulting in increased plasma concentration of LDL . Additio131I remnant ablation [p < 0.001), and increase of triglycerides from 1.98 to 2.50 mmol/L (p < 0.001), both associated with the decrease of T3 level. In this study, authors included male and female and the median age was 44. Thus, the difference result obtained in our analyses can be justified by the gender and age difference. Indeed, in a previous study, Delitala et al. showed that the relation between TSH and lipid parameters was gender-specific, and further demonstrated that the lack of estrogen could contribute to this association [A recent study reported the effect of rhTSH on 82 patients with differentiated thyroid cancer, previously treated with total thyroidectomy and ablation . The anaociation . The association , could hociation . Anyway,ociation ,31, but ociation .p = 0.066).We did not found any significant change in LDLc and HDLc. LDLc has a half-life of 3.5 days  and the 131I therapy, as demonstrated by the absence of circulating thyroglobulin. This provides an ideal model in which FT4 values remain stable during the test, due to constant LT4 supply, then analysing the direct effect of TSH alone on serum lipids. The questions whether different estrogen concentrations  might play ad additive role cannot be answered by the present study.We acknowledged some limitations of the study. First, we cannot ascertain whether blood sample have been collected in a specific phase of menstrual cycle, thus discerning possible effect of higher/different estrogen concentrations. In addition, we did not assess blood sample after the 5th day from the injection of rhTSH, and we can only hypothesise a reduction of lipid concentrations due to the progressive reduction of TSH. However, our study included patients without any residual of the thyroid gland due to surgery and We demonstrated that rhTSH can increase total cholesterol, triglycerides, and nonHDLc, independently from FT4 level. The understanding of the exact mechanism(s) through which TSH can stimulate hepatic cell to produce lipids will require additional studies.


The human respiratory syncytial virus (hRSV) and human Metapneumovirus (hMPV) are two of the leading etiological agents of acute lower respiratory tract infections, which constitute the main cause of mortality in infants. However, there are currently approved vaccines for neither hRSV nor hMPV. Moreover, despite the similarity between the pathology caused by both viruses, the immune response elicited by the host is different in each case. In this review, we discuss how dendritic cells, alveolar macrophages, neutrophils, eosinophils, natural killer cells, innate lymphoid cells, and the complement system regulate both pathogenesis and the resolution of hRSV and hMPV infections. The roles that these cells play during infections by either of these viruses will help us to better understand the illnesses they cause. We also discuss several controversial findings, relative to some of these innate immune components. To better understand the inflammation in the lungs, the role of the respiratory epithelium in the recruitment of innate immune cells is briefly discussed. Finally, we review the main prophylactic strategies and current vaccine candidates against both hRSV and hMPV. The human respiratory syncytial virus (hRSV), recently renamed human orthopneumovirus, is the leading viral agent that causes acute lower respiratory tract infections (ALRTIs), making this virus the main etiological viral agent by which infants under the age of five die ,2,3. It Infection by hRSV affects young children more severely, as well as the immunocompromised and the elderly ,7. InfecEven though hRSV was first identified over 60 years ago and has been widely studied ever since its discovery, no safe and effective vaccine has been approved to prevent the disease caused by this virus ,16.The human Metapneumovirus (hMPV) is another major viral agent that causes ALRTIs, making it an important pathogen that contributes significantly to the mortality of infants under the age of five ,2. A rephMPV infections cause more complications to young children, the immunocompromised, and elderly patients . hMPV inhMPV was discovered more recently than hRSV in 2001, and similar to the latter, no vaccine against hMPV has been approved yet ,23.Even though both of these viruses are closely related and share similar symptoms, infection with hRSV causes more cases of bronchiolitis and fewer cases of pneumonia as compared to hMPV . MoreoveIn this article, we will discuss the general characteristics of these viruses along with the innate immune response that these pathogens induce in the host. This work will be focused on dendritic cells (DCs), alveolar macrophages (AMs), neutrophils, eosinophils, natural killer (NK) cells, innate lymphoid cells (ILCs), and the complement system. Additionally, we will discuss how this response can contribute to pathogenesis and/or disease resolution. How the pulmonary epithelium can contribute to both pathogenesis and the recruitment of these immune cells will also be addressed. Current and prospective prophylactic approaches, such as vaccines and monoclonal antibodies that can help to control the infection caused by these viruses, will also be considered. Lastly, we will discuss the similarities and the differences in the immune responses elicited by hRSV and hMPV infections.hRSV and hMPV belong to the same taxonomical family of viruses, but due to individual characteristics in their genomes, they are classified in different genera within this family. In the following section, the general characteristics of these viruses regarding their genetics and life cycles will be discussed.Pneumoviridae family, Orthopneumovirus genus [m2 gene [hRSV is an enveloped, single-stranded, non-segmented, negative-sensed RNA virus that belongs to the us genus . Its genus genus . It is n[m2 gene .The envelope of hRSV contains three proteins on the surface: the glycoprotein (G), the fusion protein (F), and the short hydrophobic protein (SH). The G protein is a heavily glycosylated glycoprotein involved in the attachment of the virus to the target cell via the binding of heparin and/or annexin II on the cell surface ,27. As fThe genome of hRSV is associated with the nucleoprotein (N), the phosphoprotein (P), and the viral RNA-dependent RNA polymerase (L) to form the ribonucleoprotein complexes (RNPs). The main functions of the N protein are to coat the viral RNA in a left-handed helical nucleocapsid to protect it from mechanical, chemical, and physical damage ,37, and Matrix proteins M and M2-1 are also present in the virion as structural components ,47. The Lastly, hRSV possesses two non-structural proteins, NS1 and NS2, which are expressed in the early stages of replication. These proteins are considered to be major virulence factors of hRSV since they play an important role in the inhibition of type I IFN expression, thus promoting viral replication and spread to neighboring cells ,51,52.hRSV is able to infect bronchial respiratory epithelia. Interestingly, it has been shown that it can also infect neurons in vitro ,53, as wInterestingly, SH protein has been shown to act as a viroporin, assembling into pentameric pores in infected cells ,58, thusLastly, non-structural proteins 1 and 2 (NS1 and NS2) are encoded by the first two genes of hRSV and play crucial roles as virulence factors in early time points of infection . Both ofMetapneumovirus of the family Pneumoviridae [m2 gene, which codes for two different proteins. M2-1 and M2-2 are the result of the overlapping of two ORFs located in the mRNA from the m2 gene [hMPV is an enveloped, non-segmented, single-stranded, and negative-sensed RNA virus, which belongs to the genus oviridae ,64. The The hMPV virion consists of an envelope that has three glycoproteins on the surface: the small hydrophobic protein (SH), the attachment protein (G) and the fusion protein (F). The SH protein is a transmembrane protein of type II, which is capable of inhibiting the transcriptional activity of NF-κB, resulting in a decrease in the transcription of genes that use this pathway ,66. It hThese glycoproteins found on the surface are associated with the matrix protein (M), which is also connected with the inner membrane from the surface. The M protein interacts with various components from the virus and the cells, promoting the assembly and budding of the virus . AdditiohMPV RNA is united with the nucleoprotein (N), the phosphoprotein (P), the large polymerase protein (L), the putative transcription factor (M2-1), and the RNA synthesis regulatory factor (M2-2), forming the nucleocapsid. The association between two domains from the N protein and the virus RNA causes the helical nucleocapsid form and protects the genome from any damage ,72. The Ciliated epithelial cells are the primary target of the hMPV infection, and then the virus spreads to all the cells of the respiratory tract where viral replication takes place . The repOnce the virus arrives at the airway epithelia, the F and G proteins promote the attachment to the airway epithelial cell (AEC), where there is an interaction between the F protein and αvβ1 integrin, along with an interaction between the G protein and GAGs ,76,77. WhMPV exhibits two phases of replication, characterized by their kinetics of infection: an acute phase of infection with a peak of viral titer 7 days post-infection (d.p.i.) and a second phase of infection with a peak of viral titer at 14 or 28 d.p.i., depending on the virus strain. Of note, the genetic material of hMPV can be found up to 180 d.p.i. within the lungs of BALB/c mice .The innate immune response is the first line of defense against virtually all kinds of pathogens, including those of viral natures. Within this complex response, innate immunity plays a key role in the control of infections given its fast and antigen non-specific responses against pathogens. The most relevant innate immune response components in the contexts of hRSV and hMPV infections are neutrophils, eosinophils, macrophages, dendritic cells, natural killer cells, and the complement system. How each of these cells are activated and the immune pathology, or contribution for disease resolution that results from their recruitment, is discussed below.Dendritic cells (DCs) are considered part of the first line of defense against viruses and, most importantly, are considered to orchestrate both the innate and the adaptive immune response . DCs canIfna6gfp/+ mouse strain to define which immune cells contributed to IFN-I secretion and validated their results through the quantitation of Ifna5 and Ifnb mRNA expression by quantitative RT-PCR on FACS-sorted populations of immune cells. Considering that neither of the approaches of these authors considered direct IFN-α protein recognition and that mice possessed 14 genes coding for IFN-α [During a hRSV infection, all DCs subtypes have been shown to migrate to the lung as early as 2 d.p.i. ,88, and or IFN-α , DCs mayDCs are susceptible to hRSV infection ,93,94,95H2-polarizing and inflammatory cytokines, including TNF-α, IL-1β, IL-6, and low quantities of IL-10 has been observed, as shown in + T cells [hRSV infection induces DC maturation, as observed by the upregulation of markers, such as CD40, B7.1 (CD80), CD83, B7.2 (CD86), MHC-I, and MHC-II ,94,100,  T cells ,100. Sur T cells ,55.H2 polarization is key for hRSV pathogenesis and is largely influenced by DCs. It has been shown that the cDC2 subset is capable of polarizing T cell responses in an IL-4Rα-dependent manner, which induces poor DC maturation and TH2 bias [H2-biased adaptive response [H2-polarizing profile [During hRSV infection, poor or inadequate activation of T cells by DCs is not uncommon ,100. TH2TH2 bias . Moreoveresponse , and the profile .+ T cells to a TH2 profile [+ T cells [+ T cell activation [As previously mentioned, cDCs are of myeloid origin and both subtypes are present in the lungs during hRSV infection , and in  profile . On the  T cells ,113,114, T cells ,97. The  T cells ,97, and tivation ,94, as ptivation ,97.On the other hand, pDCs are of mixed ontogeny and are characterized by their plasmacytoid appearance and vast secretion of IFN-I. These cells contribute greatly to disease resolution, lowering viral titers, and ameliorating pulmonary inflammation and airway obstruction ,115,116.Even though there is one report stating the opposite , the con+ T cell responses [+ T cell antiviral responses [Although pDCs do not interfere with CD4esponses ,119, theesponses ,117,119.esponses ,108, whiesponses .pDCs are susceptible to hRSV infection, albeit abortive and very scarce . Infecti+ T cell, which limits airway inflammation [Studies using neonate models of hRSV infection are extremely relevant, considering that newborns and children less than two years of age are an important high-risk population. Murine neonate models of infection have revealed that neonate pDCs are capable of processing and presenting antigens, but possess an intrinsic insufficient IFN-I response in comparison to mature adult pDCs . Similarammation . Furtherammation .+ T cell responses to adult cDC1s, giving rise to an immature-like immunodominant hierarchy among CD8+ T cells [+ T cell populations that recognize particular epitopes derived from hRSV antigens. The pattern of epitope dominance is relevant in the context of infection since it shapes the adaptive immune response and defines the epitopes from an antigen that will induce a more potent immune response [high cDC1s—but not CD103low cDC1s—can establish the same immunodominant CD8+ T cell hierarchy that is observed in adult cDC1-mediated CD8+ T cell responses [+ T cells [Interestingly, lung migratory cDC1s from newborns establish different antigen-specific CD8 T cells . Brieflyresponse ,124,125.esponses . This su T cells .To date, there are no clinical reports that describe the role of DCs in the pathology induced by the infection with hMPV . However+ cDC1 subset decreases during the first weeks after the infection, but 8 weeks after the infection it reaches its normal amount [During the acute phase of infection with hMPV, DCs comprise the main immune cell population that senses this virus, along with macrophages and AECs ,128. As l amount . The hMPl amount .+ T cell activation and, as a consequence, it reduces the TH1 response [In vitro studies demonstrated that monocyte-derived DCs (moDCs) were capable of being infected by hMPV, but it was an unproductive infection . The effresponse . This coresponse ,131.When moDCs are infected with hMPV they can secrete low concentrations of IL-6 and TNF-α, and when these cytokines levels are compared to non-infected cells, the difference is not significant. However, moDCs secrete significantly higher IFN-α levels compared to non-infected cells . On the As mentioned before, pDCs are an important source of IFNs that are crucial for the appropriate antiviral response. A recent report demonstrated that the SH protein of hMPV suppresses the TLR7/MyD88 pathway in pDCs, leading to the inhibition of the IFNs expression . The M2-+ T cell, since IFNAR enhances the increase in DCs in the first stage of replication [IFNs are secreted via sensing the viral RNA. However, IFNs can be secreted through the sensing of IFN by the alpha interferon receptor (IFNAR) in the cells as well. The number of DCs decline when this receptor is absent during the infection with hMPV, causing the debilitated response of CD8lication .In vitro studies have proven that the M protein can be released from the cell during the infection and can bind with DCs, internalizing this protein in a fast way. The M protein has the ability to induce the maturation of DCs and, as a consequence, the secretion of cytokines and chemokines, such as TNF, IL-1β, IL-6, IL-12p70, IL-8, and IL-10, that promote the inflammatory response ,137.H2 polarization and poor CD4+ T cell responses against hRSV, especially when DCs themselves are infected. On the other hand, pDCs are an especially important and highly regulated source of IFN-I and are critical for the establishment of an antiviral state in the lung epithelium, as well as for the establishment of a CD8+ T cell-mediated antiviral response. Lastly, cDC1 responses also appear to be vital in the establishment of a proper CD8+ T cell response, whose immunodominant hierarchy is immature in neonates. As for DCs during hMPV-infection, it seems they play a protective role against the infection of this virus. The secretion of the cytokine IL-12p40 enhances the pro-inflammatory responses, and without it the inflammation causes intense damage in the lung tissue. The infection of DCs with hMPV impairs the proper priming of naïve T cells and can inhibit the signaling of TLR7/9, reducing the secretion of IFNs in pDCs. However, IFN levels can still be secreted by the different types of DCs. A comparison of the role of DCs between hRSV and hMPV infections can be found in In conclusion, DCs play varied roles during hRSV infection. Quite importantly, they are largely responsible for the TMacrophages are within the first cells that protect the host against the viral infection, and there are two types in the lungs: the alveolar macrophages (AMs) and the interstitial macrophages (IMs) . AMs areIn the case of hRSV, AMs are crucial for the early control of infection. During a hRSV infection, they represent one of the foremost and only sources of IFN-I, contributing to the establishment of an antiviral state in neighboring cells . IFNs arMoreover, the depletion of AMs leads to a more severe disease caused by hRSV . For exaThe protective role of AMs can be at least partly explained through the previously mentioned secretion of type I IFNs, but also through the balanced and controlled secretion of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-8, upon the encounter with the live or inactivated virus, or upon AMs infected with hRSV, as shown in H2 type adaptive immune response that is not efficient for virus clearance [AMs can be infected acutely by hRSV, as indicated above ,152. Intlearance . Howeverlearance . Intereslearance . IL-6, Tlearance ,147,154.H2 responses and harmful in the context of hRSV infection. IL-33 secretion was found to be induced by TLR7 agonists, suggesting a possible mechanism for IL-33 secretion during a hRSV infection [H2 polarization [Nonetheless, some concerns have arisen regarding the possible role AMs could play in adaptive response polarization. Importantly, they can secrete IL-33 , a powernfection . Additiorization .Another controversial point is the finding that AMs can secrete considerable amounts of IL-10 at early time points of hRSV infection . ConcernIn sharp contrast to hRSV infection, AMs seem to contribute to the pathogenesis caused by hMPV, rather than protect against the infection. In the bronchoalveolar lavage (BAL) of patients infected by hMPV, macrophages are found. Along this line, a biopsy of the lungs from these patients indicated chronic inflammation and the presence of alveolar macrophages in the tissue . The chaH1/TH2 responses, especially since it promotes the secretion of IL-4, thereby stimulating the TH2 response even more [During the acute phase of infection with hMPV, macrophages are among the main immune cell population to sense hMPV ,128. Stuven more .AMs promote the spreading of hMPV though a macrophage infection-dependent mechanism, resulting in viral dissemination and the infection of the cells of the airway epithelium. Moreover, when AMs were depleted by the administration of a liposome-encapsulated clodronate suspension, hMPV infection caused a lesser decrease in the weights of infected mice, compared to non-depleted mice—the pathology score, along with the obstruction in the airways, decreased as well . Additio−/− mice demonstrated that AMs can function properly independently of IFNAR signaling, unlike IMs [AMs are one of the main sources of IL-6, TNF-α, IFN-α/β, CCL4, GM-CSF, and G-CSF in the lungs, as shown in like IMs . AdditioThe detrimental role that AMs possess might be explained by the secretion of IL-1, since this cytokine is an important enhancer of inflammation in the lungs. The secretion of IL-1β is able to generate an inflammatory response by itself and promotes the secretion of more pro-inflammatory cytokines . MoreoveH2-polarizing agents would not be unexpected, but their involvement in the development of adaptive immune response seems to be minimal [Generally speaking, the evidence points out that the activation of AMs in the lungs is a desirable response against hRSV. Their resilience against infection, their major contribution of type I IFNs, the secretion of adequate amounts of TNF-α and IL-6, and the recruitment of NK cells to the lungs are key for the early control of infection. Their possible role as T minimal . Hence,  minimal . The difNeutrophils are the most abundant type of immune cells in the bloodstream and possess important antimicrobial capacities. They are the first line of defense against many pathogens, given their notable phagocytic capacities, the release of proteolytic enzymes and other antimicrobial peptides via degranulation, and the formation of web-like structures specialized in the capture and elimination of pathogens, called neutrophil extracellular traps (NETs) .H17 CD4+ T cells along with the production of IL-8 (CXCL8) by epithelial alveolar cells are involved in the recruitment of neutrophils to the lungs, as shown in During the infection with hRSV, the excessive infiltration of neutrophils and other granulocytes are a hallmark symptom of severe disease in children . In vitrDespite their antimicrobial capacities, a recent study shows that neutrophils are neither implicated in the elimination of hRSV in the lungs nor in the recruitment of effector or memory T cells to the lungs . MoreoveHowever, there is evidence that neutrophils can contribute to pathogenesis, causing airway inflammation and tissue damage. For instance, it has been shown that hRSV is able to induce NETosis in human neutrophils in vitro and in the BAL from children with hRSV-ALRTI ,169, andAnother harmful effect of neutrophils in the context of hRSV infection is their ability to release great amounts of reactive oxygen species (ROS) in a process called oxidative burst, non-specifically oxidizing biomolecules in the surroundings of the cell. Although quite effective for controlling pathogens, it can be very harmful to the tissues of the host. Interestingly, it has been observed that the pro-inflammatory milieu elicited by hRSV is capable of promoting an oxidative burst in neutrophils in vitro . MoreoveIn vitro studies have also shown that the hRSV infection of an epithelial alveolar cell monolayer promotes neutrophil transmigration and further supports the notion of excessive neutrophil recruitment to the site of infection . More imRegarding the infection with hMPV, during a study performed in infected patients aged between 1 and 16 years, numerous neutrophils were found in their BALF. Additionally, during a biopsy of the lungs from these patients, chronic inflammation in the airways was found . Later oDuring a study performed in hMPV-infected mice, it was shown that neutrophils represented the highest percentage of the immune cells evaluated that were present in the lung infiltrate, peaking neutrophilic infiltration 1 d.p.i. and decreasing over the course of infection. This extreme amount of neutrophils found in BAL and histological samples of bronchioli and alveolar spaces correlates with the damage caused in the lungs, such as interstitial pneumonitis and alveolitis ,161. MorReports have indicated that a high presence of neutrophils during other viral infections has been shown to induce acute damage in the lungs through NETs formation and the release of ROS, such as superoxide anions, as previously mentioned ,180. MorIFN-I signaling plays a key role in the control of neutrophils, and when this signaling is absent, an increase in the recruitment of neutrophils is observed . hMPV inEven though the presence of neutrophils seems to only contribute to the pathogenesis in the lung caused by the infection of hMPV, the lack of neutrophils has shown to aggravate the symptoms of the illness, increasing the inflammation in the lungs and making the recovery period even longer . The lacPediatric patients infected with either virus exhibited similar amounts of blood neutrophils . HoweverEven though there is controversial evidence regarding the role of neutrophils in the control of a hRSV infection, the sole presence of excess neutrophils in the airways may pose a problem for the disease severity. It is important to keep in mind that the excessive cellularity found in the BAL of hRSV-ALRTI almost certainly interferes with the normal physiology of lung function and may be an important obstacle for proper alveolar gas exchange, even when not considering the characteristic production of excessive mucus elicited by hRSV infection. As for hMPV, the role of recruitment neutrophils remains controversial since some reports indicate it causes damage in the alveoli and the bronchiole, but the lack of these immune cells greatly affects the management of the inflammation, enhancing the symptoms and, as consequence, increasing the amount of time the illness lasti. A comparison of the role of neutrophils during hRSV and hMPV infections can be found in Eosinophils are granulocytes with important phagocytic capacities, usually overlooked when studying infectious diseases other than those that are helminth-related. These cells possess basic granules on their cytoplasm that contain four major classes of cationic proteins: the major basic proteins 1 and 2 (MBP-1/2), the eosinophil cationic protein (ECP), the eosinophil peroxidase (EPO), and the eosinophilic-derived neurotoxin (EDN) . These pH2-based immunity and work synergistically with mast cells on many occasions [H2 cell expansion, such as IL-2, IL-4, IL-5, IL-6, and IL-13, as well as various prostaglandins that are involved in asthma exacerbation [EDN and ECP are of particular interest since they are RNases with antiviral properties against ssRNA viruses, particularly against pneumoviruses, such as hRSV . It is accasions . Furthererbation ,191,192.erbation ,195,196.Eosinophilia is common in the BAL of children suffering from hRSV-ALRTI . There iH2-polarizing cytokine, as shown in Firstly, there is evidence that eosinophils may be detrimental to resolving hRSV infection. For instance, it has been observed that eosinophils are susceptible to hRSV infection, which leads them to secrete higher amounts of IL-6, a THowever, there is extensive evidence suggesting that eosinophils are beneficial in the control of hRSV infection and disease. One study found that the BAL of hRSV-ALRTI children with eosinophils expressing higher membrane CD11b (a marker of activated phagocytes) required less supplemental oxygen during the treatment . On the H2 polarization and not of eosinophilia, as previously thought [Lastly, some studies suggest that eosinophils may not play any role at all during hRSV infection, or at least during FI-hRSV VED. In a FI-hRSV immunized murine model, Knudson and colleagues show that eosinophils are not required for VED, and airway obstruction still occurs in mice lacking eosinophils—VED would be a consequence of T thought . However thought . Thus, eH2 adaptive immunity during hRSV infection leads to the secretion of antibodies, amongst whom IgE is almost exclusive of a TH2 profile. The secretion of IL-5 combined with IgE in the lungs might promote eosinophil migration and activation in the tissue because eosinophils possess IgE receptors. Activation leading to the degranulation of basic proteins could be harmful to the lung and may be implicated in the long-term development of asthma [Whether eosinophils are beneficial, detrimental, or just “bystanders” during a hRSV infection needs to be determined. Still, we believe eosinophilia must almost certainly be involved in the respiratory pathology of hRSV, because the cellularity of the infiltrate can interfere with the normal physiologic functions of the lungs , as discussed above for neutrophils. Moreover, the establishment of a signature Tf asthma ,214. Altf asthma ,191. We The role of eosinophils during hMPV infection is not clear, mainly because it has not been thoroughly studied. During a study performed in a patient infected with hMPV, the BAL was demonstrated to contain eosinophils while the patient was having a wheezing episode, shown in −/− or IRF3/7−/− mice developed eosinophilia during the infection with hMPV. Additionally, CCL24, a chemoattractant of eosinophils, also increased their concentration in the lungs [It was demonstrated that IRF7he lungs . Since hhe lungs ,216.H2-polarized adaptive response. IL-4 is capable of promoting the differentiation and expansion of eosinophils that can cause a TH2 profile [Even though there was a presence of eosinophils in the samples from patients infected with hMPV, studies in BALB/c mice demonstrated that eosinophilia is not a common phenomenon in this model . However profile . Based o profile .Eosinophils are more abundant in the blood of hRSV-infected patients than in the blood of hMPV-infected patients, and the same result is obtained when the samples are from NPA ,219. ThuNatural killer (NK) cells are innate lymphoid cells that mediate important antiviral responses, as well as those against intracellular bacteria and cancer cells. They have important cytotoxic capabilities mediated by perforin and granzyme, and are an important source of IFN-γ .+ T cells, its concentration in the BAL samples from the hRSV-infected mice is so low that it does not induce NK cell chemotaxis [Studies regarding the presence of NK cells in the BAL of hRSV-infected children are controversial. Some studies have found a high presence of these cells ,221, wheemotaxis . Howeveremotaxis , suggest+ T cell-depleted murine models, in which IFN-γ secretion is impaired and NK-cell derived IFN-γ is not excessive since there is no CD8+ T cell-derived IFN-γ [It has been observed that lung NK cells at early time-points of infection express activation markers and secrete high quantities of IFN-γ ,230,231.ed IFN-γ .H2 profile [+ T cells [NK cell activation and IFN-γ secretion have controversial antiviral activities. Even though NK cell activation is associated with lower viral loads , and the profile , lower l T cells , and eos T cells . Some st T cells . Thus, aThe hRSV infection of both neonate and adult NK cells has recently been reported . NK cellUnlike hRSV, the information concerning NK cells and their role in the infection with hMPV is rare, and most reports do not agree with one another after an infection with hMPV, the number of NK cells increases up to 3 d.p.i. in the lungs, and they are completely functional, as shown in + T cells and CD8+ T cells in the lungs do not vary either. According to the authors, NK cells are not essential for the clearance of the virus since their absence does not make any difference in the recruitment of immune cells [In the study where the depletion of NK cells did not change the viral load, an anti-NK1.1 antibody was used to accomplish the depletion of these cells. This antibody can produce the depletion of both the NK cells and natural killer T (NKT) cells, and this factor might affect the results ,235. In ne cells .NK cells have receptors that can inhibit or activate their state of activity, and within these receptors, the natural cytotoxicity receptors (NCRs) can be found, which are one of the classes of receptors with the most influence on the activation of NK cells. NKp46 is one of these receptors in humans, while NCR1 is its homologue in mice . These rInterestingly, there is a differential recruitment of NK cells during the infection with hRSV or hMPV. One study demonstrated that NK cells were significantly more recruited to the site of infection under hMPV than hRSV by day 4 p.i.; however, by day 7 p.i., the amounts of NK cells were similar .In conclusion, NK cells play a detrimental role during hRSV infection, promoting an inflammatory milieu that contributes to lung inflammation and promotes airway obstruction, thus contributing to the pathology associated with the disease, despite the possible contribution of NK cells to viral clearance. On the other hand, NK cells do not seem to enhance the immunopathology caused by hMPV and their protective or pathogenic capacities remain to be elucidated, although there is some evidence that suggests that NK cells contribute to viral clearance. Even though it seems that both viruses promote different NK cell responses, it is clear that the recruitment of these cells to the lungs takes place. Further insights regarding their specific roles during pneumovirus infection are needed to further comprehend the immunopathology caused by these viruses. A comparison of the roles of NK cells during hRSV and hMPV infections can be found in Innate lymphoid cells (ILCs) are cellular components of the innate immune system of lymphoid origin, which were discovered nearly a decade ago. They stem from the common lymphoid progenitor (CLP), further mature into different subsets, and migrate to mucosal tissues, poised in key positions for pathogen recognition and subsequent rapid activation .H1 hallmark transcription factor [H2-related cytokines upon activation, such as IL-4, IL-5, IL-9, IL-13, IL-25, and IL-33, and express GATA3, a TH2 hallmark transcription factor [H17-polarized CD4+ T cells [Interestingly, NK cells represent the one and only cytotoxic class of ILCs known to date and are related to other ILCs only early in development . These cn factor . ILC2s sn factor . Interesn factor ,242,243. T cells .H profiles, ILCs do not possess TCRs, do not exhibit wide genetic diversity, and are not antigen-specific [+ T cells through antigen presentation in class II MHC, which activates both the ILC and the CD4+ T cells in an IL-2-dependent fashion. This is especially true for ILC2s, which have been shown to strongly promote the TH2 responses [It is important to note that, although there are similarities between ILC subtypes and Tspecific . Howeveresponses ,241.H2 response.There are few reports regarding the role of ILCs during the hRSV infection, especially in humans. However, it has been observed that elevated levels of ILC2s in the BAL of the hRSV-infected children correlate with a higher disease severity , probablH2 response. It has been suggested that TSLP is responsible for the activation and expansion of this IL-13-producing subset, since TSLPR KO mice exhibited diminished weight loss, lower levels of IL-13-producing ILC2s, lower lung IL-13 levels, and lower airway hyperresponsiveness (AHR) compared to WT mice [H2 responses which is upregulated early in hRSV infection, is not involved in ILC2 expansion in the lung [Studies using murine models have demonstrated that a significant expansion of the ILC2 subset occurs in the lung early in hRSV infection ,247,248. WT mice . TSLP ne WT mice . Interesthe lung , but canthe lung .H2 responses. Xanthine oxidase is the enzyme responsible for the degradation of nucleic acids and contributes to uric acid production in the lungs by this pathway, which is known to induce allergic responses [It has been reported that uric acid is produced during hRSV infection and can also promote hRSV pathogenesis by enhancing TSLP and IL-33 production , which lesponses . Targetiesponses . Targetiesponses .+ T cells during hRSV infection, which allows for ILC2 expansion and activation—as measured by IL-5 and IL-13 production—in an IL-2-dependent manner [+ T cell-derived IL-2 is what activates ILC2s [+ T cell expansion, but not the IFN-γ-expressing CD4+ T cell expansion, in infected mice [+ T cell activation requires cell–cell contact and OX40–OX40L interaction [Interestingly, ILC2 activation appears to be dependent on interaction with CD4t manner . ILC2s fes ILC2s . On the ted mice . Moreoveeraction .H2 and TH17 responses [Finally, it has been observed that the expression of the transcription factor STAT1 is critical for a proper ILC response against hRSV. It has been observed that STAT1 KO mice exhibit a lower recruitment of IFN-γ-producing ILC1s during hRSV infection, as well as higher numbers of IL-5- and IL-13-producing ILC2s, and IL-17A-producing ILC3s, which are detrimental to the hRSV infection since they promote harmful Tesponses . This fuOn the other hand, there are no studies regarding these cells and their relationship with hMPV infection. It would be of interest to investigate whether these cells are affected by the infection of hMPV and if they contribute to lung damage.+ T cell activation and subsequent IL-2-dependent ILC2 activation, as well as TH2 polarization on CD4+ T cells during hRSV infection. This would make sense, given that ILCs have been observed presenting antigens on MHC-II, albeit less efficiently than DCs [H2 responses, both of which can ultimately lead to distinctive hRSV-caused eosinophilia and immunopathology.Overall, the data suggest that the antigen presentation on MHC-II by ILC2s could be the mechanism by which ILC2s induce CD4than DCs ,252. HowILC1s could possess antiviral capacities and be beneficial for hRSV infection, but studies regarding this cell type have not been performed. Similarly, studies regarding the role of ILC3s should also be performed to establish the precise role that these cells play during hRSV infection. A summary of the role of ILCs in hRSV infection can be found in The complement system is a group of soluble plasmatic proteins with intrinsic and sequential protease activities that possess cytolytic and proinflammatory properties. The cascading of protease activities culminate in the formation of a membrane attack complex (MAC) on the surface of the target cell, inducing the formation of a large pore on the cell membrane that leads to the loss of osmotic control and lysis . AdditioInitial observations during the 1980s describe complement fixation on the surface of the hRSV-infected cells in vitro, through both the classical and alternative pathways as shown in More recently, it has been observed that two anti-G hRSV neutralizing antibodies (CB002.5 and CB017.5) require the presence of the complement to neutralize hRSV infection in Vero cells . It has H17-related cytokines in the lung , as well as lower transcription levels of RORγt, without changes in TNF-α, IFN-γ, IL-4, or IL-5 levels [H17 responses during hRSV infection [However, the complement system also plays a pathogenic role during hRSV infection, mediated mainly by the anaphylatoxins C3a and C5a produced during the proteolytic cascade of complement proteins. For instance, hRSV-infected mice lacking the receptor for C3a (C3aR1) exhibit lower AHR, lower neutrophil infiltration, higher macrophage recruitment to the lung, and lower viral load than WT mice . Moreove5 levels . These dnfection . On the nfection . Moreovenfection . These dnfection . This isnfection .Consistent with its involvement in antibody-mediated immune responses, the complement system has also been proposed to play a role in FI-hRSV VED, since there is a significantly higher deposition of C3 in FI-hRSV-immunized mice during hRSV infection, as well as C4 deposition in the lungs of both patients who died during the FI-hRSV vaccine trial . SurprisConsidering that part of hRSV-induced pathology is mediated by anaphylatoxins, it is interesting to note that hRSV has been shown to induce the expression of CD59 in vitro in A549 cells . CD59 isOn the other hand, there are no studies regarding the fixation and activation of the complement system in a hMPV infection. Similarly, there are no studies regarding the role of complement-derived anaphylatoxins or the role of the complement proteins in humoral or cellular responses against hMPV. Certainly, this is an interesting and unexplored area that might provide insight into the components that mediate cellular infiltration and antibody-based responses in a hMPV infection.In conclusion, the classical pathway of the complement system mediates part of the antibody responses against hRSV and is critical for a proper humoral response. The complement system possibly promotes viral clearance through the lysis of infected cells through both classical and alternative pathways. However, the excessive or uncontrolled production of anaphylatoxins can be detrimental during hRSV infection and can promote cellular infiltration and increased AHR, as well as inefficient viral clearance. A summary of the role of the complement system in hRSV infection can be found in The respiratory epithelium is an important driver of the immune response against respiratory viruses, as these cells are implicated in the secretion of large quantities of chemokines upon infection. This will attract immune cells to the site of infection, as well as other various cytokines that modulate the immune response .H2 adaptive immune response. Many of these studies are based on in vitro models, and we only discuss the secretion of cytokines that have been observed in at least one type of human primary cell culture [For hRSV, epithelial cells have been seen to contribute to the promotion of local inflammation and angiogenesis, the recruitment of various inflammatory cell types, and the development of a harmful T culture ,297,298.Regarding acute local inflammation, the hRSV-infected epithelial cells have been shown to secrete IL-1α/β, IL-6, HMGB1, TNF-α, VEGF, and FGF ,274,275.Epithelial cells are deeply implicated in the recruitment of many immune cell types, among which neutrophils, monocytes, eosinophils, and NK cells are found. The extensive secretion of neutrophil chemoattractant CXCL8 (IL-8) has been found to occur during the infection with hRSV as early as 2 h.p.i. and to be steadily secreted for as long as 6 d.p.i. ,273,276.H2-polarizing cytokines by 24 h.p.i., such as TSLP, CCL2 (MCP-1), MCP, HMGB-1, IL-25, and IL-33 [+ T cells via the Jagged/Notch-1 contact-dependent axis, directly influencing TH2 differentiation in vitro [Finally, hRSV infected epithelial cells have been shown to secrete Tnd IL-33 ,286,287.nd IL-33 . Moreovein vitro .H2 response against infection due to the secretion of TSLP and IL-33 [H2 cytokine response through DCs [H2 response and it is known to contribute to the damage produced in the lungs [Regarding the hMPV infection of AECs, it seems to induce a Tnd IL-33 . TSLP isough DCs ,299. Eveough DCs . As for he lungs .The hMPV-infection of epithelial cells from the lung causes the secretion of the chemokines IL-8 (CXCL8) and CCL5 (RANTES). IL-8 can be detected as early as 6 h.p.i. while CCL5 can be observed after 12 h.p.i., although both cytokines can achieve the highest secretion at 48 h.p.i. . The secCytokines IL-6, CXCL10 (IP-10) and CCL2 (MCP-1) were also found to be secreted by epithelial cells infected by hMPV. IL-6 reaches the highest concentration 48 h.p.i., while CXCL10 and CCL2 reach their peaks 24 h.p.i decreasing their levels at 48 h.p.i. Even though CCL3 (MIP-1α) and CCL4 (MIP-1β) secretions are higher compared to non-infected controls, their levels remain low . It has Lastly, the infection with hMPV is able to induce the secretion of IFN-α/β, and this secretion can activate IFN-signaling from other epithelial pulmonary cells. However, the amounts of epithelial cell-derived IFN-α are lower compared to those derived from moDCs . AdditioH2-polarizing cytokines, which overall lead to a poor response against hRSV. As for the hMPV-infection, it seems that most of the cytokines secreted by AECs promote a TH2 inflammatory response, contributing to the obstruction and damage of the tissue. However, the expression of IFN-λ contributes to the protection of the pulmonary epithelium. A comparison between cytokine secretions from the RSV- and hMPV-infected epithelial cells can be found in The immunopathology associated with hRSV infection is largely mediated by the secretion of inflammatory cytokines and various chemokines that lead to neutrophilia and eosinophilia, both hallmark symptoms of the hRSV infection, and the secretion of TVaccines are a great—if not the best—way of preventing virus-caused illness. However, neither hRSV nor hMPV have approved vaccines yet . Many prIn the case of hRSV, there are a few monoclonal antibodies and several vaccine candidates with prophylactic potential. These are shown in Palivizumab is a F-hRSV-targeting monoclonal antibody that is currently on the market and represents the main prophylactic approach against hRSV. It has been shown to be protective and to effectively neutralize hRSV ,306,307.Motavizumab is an optimized variation of palivizumab, obtained by fine-tuning the amino acids present in the complementarity determining region of the antibody . AlthougOther strategies involving monoclonal antibodies are MEDI8897, a F-targeting antibody currently ongoing a phase 3 clinical trial ,314, andRegarding vaccination strategies against hRSV, there are many candidates currently ongoing clinical trials . HoweverMany live-attenuated vaccines are currently ongoing phase 1 clinical trials. These vaccines consist of deletion-bearing hRSV strains . The mosH1 response [Mycobacterium tuberculosis in immunized newborns.A chimeric BCG-vectored vaccine expressing the N protein of hRSV was recently completed in a phase I clinical trial (data not shown). The rationale behind this formulation is supported by the fact that the N protein is expressed on the membranes of infected cells, thus exposing this antigen ,55. Interesponse ,320. AnoVector-based vaccines have also been developed and are currently ongoing phase 2 clinical trials. Three of these vaccine candidates are based on the adenovirus-mediated delivery of antigens and one of them is based on a modified vaccinia Ankara virus. All are in ongoing phase 2 clinical trials. These vaccines rely on poorly replicating vectors and induce protective humoral and cellular immunity against hRSV antigens—the F protein in particular .Particle vaccine candidates are being developed and are in ongoing clinical trials for three target populations: pediatric, elderly, and maternal. They are based on the delivery of stabilized post-F antigens in the form of nanoparticles, and are among the most promising candidates .Finally, there are many subunit vaccine candidates, although none of them are directed at the pediatric population, given the history of ERD with subunit vaccines. These vaccines encompass SH, G, or F delivery and include a variety of adjuvants in their formulation .In the case of hMPV, there are no vaccines or antibody treatments approved yet to treat or prevent the infection, but there are a few monoclonal antibodies and vaccine candidates with prophylactic potential ,338. TheA vaccine candidate—that recently finished its phase 1 clinical trials—is a live attenuated recombinant hMPV expressing the P protein from the avian metapneumovirus (rHMPV-Pa). In this study, the vaccine was tested in infants that resulted in being seronegative for hMPV, indicating that this vaccine candidate was not immunogenic enough in this cohort . AnotherAdditionally, it is important to mention that vaccine candidates against hMPV are continually being proposed. Among these highlights, it can be found a recombinant BCG expressing the P protein of hMPV (rBCG-P), which so far has demonstrated—in pre-clinical models—that it promotes viral clearance without inducing tissue damage .As for prophylactic approaches against hMPV, mAbs are a good way to protect against this virus and to reduce the most severe symptoms. The antibody 54G10 is a human mAb which was compared to palivizumab, as it targets the F protein of hMPV. This mAb was tested in a mice model and the results demonstrated a significant neutralizing effect along with inducing a reduction in the viral titers in the lungs ,342. EveThe innate immune response against respiratory viral pathogens can either be protective or contribute to damage against the lung epithelium. Even though hRSV and hMPV come from the same virus family and share extensive genomic similarities, along with having similar entry mechanisms into epithelial cells, they produce different innate immune responses.H2-promoting cDCs. The roles of neutrophils and eosinophils are controversial, due to some reports suggesting they have a protective antiviral role, while others propose they contribute to lung damage. Finally, NK cells, ILC2s, and the excessive activation of the complement system contribute to the pathogenesis of hRSV through uncontrolled inflammatory mechanisms. The respiratory epithelium is involved in the recruitment of innate immune cells, which contribute to the symptoms of hRSV infection. On the other hand, the hMPV infection leads to the recruitment of DCs, AMs, neutrophils, eosinophils, and NK cells. Among these cells, DCs have a protective role during hMPV infection. On the contrary, AMs contribute to damage caused in the lung epithelium. The role of neutrophils is controversial due to some reports suggesting that they have a protective role, while others propose they contribute to lung damage. Lastly, the protective role of eosinophils and NK cells remains to be studied, along with the possible contribution to the inflammation that ILCs and the complement system might have, which could be induced during the infection. The respiratory epithelium plays a role in the recruitment of these innate immune cells, contributing to the symptoms of hMPV infection.The hRSV infection leads to the recruitment of DCs, AMs, neutrophils, eosinophils, NK cells, ILCs, and the activation of the complement system. Among these cells, AMs play a protective role against the infection with hRSV, while DCs can be protective—as seen for IFN-I-secreting pDCs—or detrimental, especially when infected by hRSV—as seen for TThe continuous efforts to develop a vaccine that can protect the population at risk against these viruses are important, in order to prevent at-risk groups from getting sick or generating severe symptoms caused by the immune response against these respiratory viruses. Certainly, an adequate vaccine candidate should elicit a proper innate immune response that can balance out the excessive inflammation that characterizes infection by these viruses, while still inducing protective and adequate adaptive immune responses.