Objectives: Respiratory syncytial virus (RSV) causes lower respiratory tract infection (bronchiol... more Objectives: Respiratory syncytial virus (RSV) causes lower respiratory tract infection (bronchiolitis) in newborns/infants directed by the mononuclear cell accumulation to the lungs. Undeveloped immune system in infants is considered to be cause of severe form of illness, although RSV itself has potential to skew the effective antiviral type-1 immune reaction to less effective type-2. Type-1 related chemokines (IP-10, MIG and fractalkine) predominantly induce migration of CXCR3 and CX3CR1 positive, type-1 profiled lymphocytes that are necessary to control viral infections. Opposing type-2 related chemokines (TARC and MDC) are responsible for migration of CCR4-positive type-2 lymphocytes involved in allergic immune responses. We postulate that RSV-infected infants produce elevated type-2 vs. type-1 cytokine-associated chemokines that compromise effective antiviral response. Methods: PMBC were isolated from blood samples of RSV-infected hospitalized infants (N=14), 6 infants infected with influenza and adenovirus, 9 age-matched healthy controls and from blood samples of 10 infants collected 4-6 weeks after first sampling. PBMC were simultaneously stained with mAb´s for chemokine receptors (CCR4, CXCR3, CX3CR1), and lymphocyte subpopulation markers. Multiparametric analyis was performed on LSRII flow cytometer. Frozen serum samples were used to measure soluble chemokine (TARC, MDC, IP-10, MIG, fractalkline, I-TAC, TSLP) concentration. Results: In acute phase of RSV- and other viruse-infected infants, the predominant chemokine in serum is IP-10. TARC is overproduced in RSV while fractalkine and MIG are predominant in acute influenza and adenovirus infections. Interestingly, 4-6 weeks after first sampling, serum levels of TARC, MDC, IP-10 and MIG increased even more in RSV-infected infants. In acute phase, higher percentages of B-lymphocytes and CTL's express CCR4, CXCR3 and CX3CR1 compared to healthy and other-viruses infected infants. In convalescent phase, percentages of CCR4-positive B-lymphocytes and CTL's didn't change while there was increase of CXCR3 expression. Conclusion: Mixed type of cytokine production during acute phase of RSV infection could be observed on both the chemokine and chemokine receptors level. Higher TARC concentration imposes type-2 immune reaction at inflammatory site that could lead to more pronounced RSV-driven inflammatory process with severe clinical findings since CCR4-positive lymphocytes contribute to type-2 cytokine synthesis and confront effective type-1 cytokine-driven antiviral immune reaction.
Acute RSV infection in infancy produces some asthma-like symptoms and may be followed by a recurr... more Acute RSV infection in infancy produces some asthma-like symptoms and may be followed by a recurrent wheeze later in the childhood. It has been proposed that RSV infection stimulates type 2 cytokine responses, akin to those found in atopy and asthma. We obtained peripheral blood cells from RSV-infected infants (n=30) and healthy controls (n=10). After restimulating them in vitro, intracellular IL-4 and IFN-g were measured by flow cytometry. Cells from RSV-infected infants produced more IL-4 and less IFN-g than healthy controls. Interleukin-4 production was more frequent in CD8 than CD4 cells, and the bias towards IL-4 production was the greatest in infants with mild infections. Suprisingly, B-lymphocytes in peripheral blood produced IL-4 as well. Our conclusions are that RSV infection is associated with IL-4 production in peripheral T cells, and that peripheral blood in infants with severe disease may be depleted of cytokine-producing cells.
Sažetak: Toll-like receptors (TLRs) are a family of germ-line encoded proteins involved in host d... more Sažetak: Toll-like receptors (TLRs) are a family of germ-line encoded proteins involved in host defense by their ability to recognize conserved components of different microorganisms. According to current data, TLRs that recognize bacterial and fungal cell ...
Respiratory syncytial virus (RSV) causes acute respiratory tract illness across all ages while lo... more Respiratory syncytial virus (RSV) causes acute respiratory tract illness across all ages while lower respiratory tract infection (LRTI) is usually a result of primary infection in infants. Supportive care is the mainstay of therapy and includes oxygenotherapy, use of bronchodilators and corticosteroids, although no convincing data exist for their efficacy. Antimicrobial innate defense along with transferred maternal immunoglobulins play a significant role in infants while immune system undergoes conditional maturation. Beta- defensins are small antimicrobial peptides produced by epithelial cells and mononuclear leukocytes with wide pathogen specificity, displaying neutralization/inactivation capability. Even more, premature newborns show lower beta- defensin 1 production that might compromise early innate response. We determined levels of beta-defensin 1 and 2, IL- 1beta, TNF-alpha and IL-6 in BAL samples from infants with RSV-caused LRTI and infants with bacterial pneumonia in order to determine a profile of secreted antimicrobials. Infants admitted to ICU with confirmed RSV- infection (N=13) and infants with bacterial pneumonia (N=8) underwent to bronchoscopy. Infants with human metapneumo virus (HMPV) infection (N=3) were also included. High levels of both beta- defensins were detectable in majority of infants with higher levels in infants with acute RSV infection compared to infants with bacterial pneumonia and HMPV infection. Beta-defensin levels correlated with levels of pro-inflammatory TNF- alpha and IL- 1beta. Although, RSV and bacteria could both stimulate secretion of beta-defensins via pro- inflammatory cytokine loop, higher levels of beta- defensins 1 and 2 in acute RSV infection. Sustained high level of antimicrobial production by RSV challenged epithelial cells and infiltrated leukocytes could augment local inflammation and emphasize immunopathology seen in RSV infection.
Nije razjasnjeno znacenje P-glikoproteina (Pgp), biljega visestruke otpornosti na lijekove (engl.... more Nije razjasnjeno znacenje P-glikoproteina (Pgp), biljega visestruke otpornosti na lijekove (engl. multidrug resistance, MDR) u kronicnoj B-limfocitnoj leukemiji (B-CLL). Cilj istraživanja bio je odrediti da li MDR-status pri dijagnozi bolesti i tijekom lijecenja korelira s klinickim pokazateljima i ishodom lijecenja. Stoga smo određivali razinu Pgp-a u perifernoj krvi 42 bolesnika s B-CLL-om i 36 zdravih dobrovoljaca koristeci protocni citometar i monoklonska antitijela (mAt) na intracelularne (C-219) i ekstracelularne (MRK-16) epitope Pgp-a. Rezultati su izraženi kao omjer intenziteta fluorescencija mAt i izotipske kontrole (engl. ratio of mean of fluorescences, RMF). Pokazano je da su u zdravim ispitanicima kako intra tako i ekstracelularni biljezi Pgp-a znacajno (p<0.001) poviseni u starijih osoba. U nelijecenih bolesnika s B-CLL-om nađene su vise vrijednosti za RMF (medijani C-219=3.1, MRK-16=2, 9) u odnosu na lijecene (1, 9 odn. 2, 0). Ove vrijednosti nisu korelirale s klinickim pokazateljima (TTM ili RAI-status). Nadalje smo pratili bolesnike koji su primali terapiju s MDR-neovisnim lijekovima (klorambucil ili FAMP). U bolesnika koji su odgovorili na lijecenje (kompletna ili djelomicna remisija) pokazan je znacajan pad vrijednosti za RMF (C-219 sa 6, 8 na 2, 4, a za MRK-16 s 3, 0 na 1, 4 ). U bolesnika koji nisu odgovorili na terapiju vrijednosti Pgp-a su porasle ili se nisu mijenjale. Nasi rezultati upucuju da ekspresija Pgp-a korelira s terapijskim odgovorom cak i u lijekova ciji mehanizam djelovanja nije ovisan o Pgp-u.
Background: IaIp are group of serine proteases inhibitors. They are important in vivo modulators ... more Background: IaIp are group of serine proteases inhibitors. They are important in vivo modulators of endogenous proteases including trypsin, human leukocyte elastase, plasmin and cathepsin G. Release of endogenous proteases plays an important role in inflammation, sepsis, wound healing and metastasis. A significant decrease of plasma IaIp levels occurs in adult and newborn sepsis. Our previous animal studies using the polymicrobial sepsis rat (adult) model of cecal ligation and puncture demonstrated the beneficial effects of intravenous IaIp administration in improving morbidity and mortality. Objective: The aim of this study is to evaluate the effects of parenterally administered IaIp in an in vivo animal model of neonatal sepsis. Design/Methods: Sepsis was induced in 2-3 days old Fischer rats with a subcutaneous injection of E. Coli (BORT). A bacterial dose response curve of lethality was determined. 2 CFU/gram body weight was lethal at 40-50 hours after the infection. In the subsequent experiment, three day old animals (n=8) were injected with this dose of E. Coli and then randomized into two groups. The treatment group (n=4) received three intraperitoneal injections of 250 micrograms of purified human IaIp, at 6, 12 and 24 hours after the infection. The control group received equal amount of human albumin at the same time intervals. Results: All pups in the control group died within 40-44 hour after the infection. Animals in the treatment group survived up to 78 hours. Conclusions: These results suggest that IaIp offers a beneficial effect in septic newborn rats and warrant further investigation. Questions being pursued include: optimal dose vs. response to IaIp, optimal timing of IaIp administration, pharmacokinetics of IaIp, effectiveness of IaIp in other models of sepsis (E.g. Group B strep, LPS).
Respiratory syncytial virus (RSV) is the most common seasonal viral pathogen in children by secon... more Respiratory syncytial virus (RSV) is the most common seasonal viral pathogen in children by second year of life and in some cases it causes a severe respiratory disease. Frequent RSV reinfections and similarity of clinical findings with allergic asthma (airway hyper-responsiveness, wheezing) indicate development of mixed type- 1/type-2 immune response. Presence of IFN-γ/IL-4 in acute RSV infection could be confirmed by simultaneous production of type-1 associated (CXCL10) and type-2 associated (CCL17) chemokines, responsible for recruitment of activated CXCR3+ and CCR4+ T cells detected at higher percentages in the periphery of diseased infants indicating inadequate immune response. As CCR4 is not type-2 exclusive chemokine receptor, and could be found on different T cell types, addition of CRTh2 chemokine receptor analysis would contribute to more accurate type-2 immune response assessment. Moreover, most of conclusions regarding aberrant type of anti-RSV immune response in humans are extrapolated from peripheral blood analysis, and acquisition of bronchoalveolar lavage (BAL) samples of infected infants would provide unique insight into development of local immune response. BAL samples were obtained from infants hospitalized due to sever bronchiolitis or pneumonia caused by RSV (n=14) or other respiratory viruses (n=8) and bacterial pathogens (n=7) where bronchoscopy was clinically ascribed. Concentrations of chemokines CXCL10 and CCL17 were assessed using commercially available ELISA kits. Isolated cells were immunophenotyped for CD4 and CD8 T cell specific markers and specific chemokine receptors CXCR3, CCR4 and CRTh2. Cells were analysed on LSRII flow cytometer and data analysed using Statistica v7.0 software. BAL samples of all infected infants were found positive only for CXCL10, while CCL17 was undetectable regardless of pathogen. RSV infected infants had higher percentage CXCR3+ CD4 and CD8 T cells compared to other viral respiratory infections. High percentages of CCR4+ CD4 and CD8 T cells were also detected in RSV infected infants. Coexpression of CRTh2 chemokine receptor was detected on CCR4+ CD4 and CD8 T cells in RSV infected infants. The presence of type-2 immune response in lungs of RSV infected infants was confirmed in regards of higher expression of CCR4 and CRTh2 chemokine receptors on CD4 and CD8 T cells. Although CCL17 was not detected at the time of bronchoscopy, a high CXCL10 concentration implicates efficient tracking of antiviral and pro-inflammatory CXCR3+ T cell to the lungs. As CCL17 and CCR4 are, beside type-2 T cells, also involved in regulatory T cell (Treg) migration, undetectable CCL17 in RSV infected infants could explain more severe disease due to insufficient number of Treg cells in affected tissue.
Innate immunity is important part of immune system with ability to recognize and act (neutralize)... more Innate immunity is important part of immune system with ability to recognize and act (neutralize) upon pathogen challenge. Infants are particularly sensitive to different viral infections with emphasized detrimental effect of respiratory syncytial virus (RSV) induced recurrent infections in infancy related to inadequate anti-viral immunity post-infection. Toll-like receptors (TLRs) are part of the immune system able to recognize and activate immune system upon pathogen challenge. TLR3, 7, 8 recognize genomic viral RNA molecules initiating antiviral control mechanisms. Possible explanation for frequent RSV re-infections in infancy could be inadequate TLR3, 7, 8 engagement in innate immunity responses resulting in lower cytokines synthesis and dendritic cell maturation, due to lower TLRs expression in peripheral blood lymphocytes. In our study specific monoclonal antibodies against human TLR3, 7, 8 molecules were used to determine expression profile of peripheral blood leukocyte subpopulations from healthy and RSV-infected infants by flow cytometry. We have found that RSV-infected infants had lower percentages of TLR3-expressing monocytes, NK cells, T and B cells than healthy controls. Although expression of TLR7 in different leukocyte populations didn’ t statistically differ between healthy and infected infants, a tendency of lower percentages of TLR7 in monocytes, T and B cells was observed. The TLR8, cognate of TLR7 molecule, is expressed on smaller percentage of NK and T cells in RSV infected infants. The comparison of TLR7 and TLR8 expression between different age groups revealed lower expression in monocytes from peripheral blood of infants (diseased and healthy group) than in monocytes from healthy adults and newborns. In conclusion, lower expression of TLR3, 7, 8 stresses out inadequate early antiviral and innate immune mechanisms responsible for the RSV replication control and generating an effective immune response.
Toll-like receptors (TLRs) are part of the innate immune system able to recognize pathogen-associ... more Toll-like receptors (TLRs) are part of the innate immune system able to recognize pathogen-associated molecular patterns and activate immune system upon pathogen challenge. TLR7 and TLR8 recognize single-stranded viral RNA and initiate antiviral control mechanisms. A RNA virus particularly detrimental in infancy is respiratory syncytial virus (RSV). RSV can cause severe lower respiratory tract disease and recurrent infections related to inadequate development of anti-viral immunity. The reason for frequent RSV re-infections in infancy could be inadequate TLR7 and TLR8 engagement by viral RNA resulting in lower synthesis of anti-inflammatory mediators and antigen presentation, possibly due to a lower TLR expression in peripheral blood mononuclear cells (PBMC) of a still developing immune system. We determined TLR7 and TLR8 expression in PBMC subsets from infants with primary RSV infection and their healthy controls using multi-parameter flow cytometry. We found the expression of TLR7 and TLR8 in monocytes lower in infected infants than in healthy controls. Also, the comparison of TLR7 and TLR8 expression between different age groups revealed that they are expressed at lower level in monocytes from infants (both diseased and healthy) when compared to healthy adults and newborns. Unexpectedly, TLR8 in T cells and NK cells was detected in lower percentage in infected infants when compared with healthy controls. Difference in percentage of both cell subsets positive for TLR8 between healthy and infected infants emphasize possible role in mounting anti-RSV immunoreactions. In conclusion, difference in TLR7 and TLR8 expression stresses out their importance in early antiviral immune mechanisms responsible for the control of RSV replication and generation of an adaptive immune response.
Objectives: Respiratory syncytial virus (RSV) causes lower respiratory tract infection (bronchiol... more Objectives: Respiratory syncytial virus (RSV) causes lower respiratory tract infection (bronchiolitis) in newborns/infants directed by the mononuclear cell accumulation to the lungs. Undeveloped immune system in infants is considered to be cause of severe form of illness, although RSV itself has potential to skew the effective antiviral type-1 immune reaction to less effective type-2. Type-1 related chemokines (IP-10, MIG and fractalkine) predominantly induce migration of CXCR3 and CX3CR1 positive, type-1 profiled lymphocytes that are necessary to control viral infections. Opposing type-2 related chemokines (TARC and MDC) are responsible for migration of CCR4-positive type-2 lymphocytes involved in allergic immune responses. We postulate that RSV-infected infants produce elevated type-2 vs. type-1 cytokine-associated chemokines that compromise effective antiviral response. Methods: PMBC were isolated from blood samples of RSV-infected hospitalized infants (N=14), 6 infants infected with influenza and adenovirus, 9 age-matched healthy controls and from blood samples of 10 infants collected 4-6 weeks after first sampling. PBMC were simultaneously stained with mAb´s for chemokine receptors (CCR4, CXCR3, CX3CR1), and lymphocyte subpopulation markers. Multiparametric analyis was performed on LSRII flow cytometer. Frozen serum samples were used to measure soluble chemokine (TARC, MDC, IP-10, MIG, fractalkline, I-TAC, TSLP) concentration. Results: In acute phase of RSV- and other viruse-infected infants, the predominant chemokine in serum is IP-10. TARC is overproduced in RSV while fractalkine and MIG are predominant in acute influenza and adenovirus infections. Interestingly, 4-6 weeks after first sampling, serum levels of TARC, MDC, IP-10 and MIG increased even more in RSV-infected infants. In acute phase, higher percentages of B-lymphocytes and CTL's express CCR4, CXCR3 and CX3CR1 compared to healthy and other-viruses infected infants. In convalescent phase, percentages of CCR4-positive B-lymphocytes and CTL's didn't change while there was increase of CXCR3 expression. Conclusion: Mixed type of cytokine production during acute phase of RSV infection could be observed on both the chemokine and chemokine receptors level. Higher TARC concentration imposes type-2 immune reaction at inflammatory site that could lead to more pronounced RSV-driven inflammatory process with severe clinical findings since CCR4-positive lymphocytes contribute to type-2 cytokine synthesis and confront effective type-1 cytokine-driven antiviral immune reaction.
Acute RSV infection in infancy produces some asthma-like symptoms and may be followed by a recurr... more Acute RSV infection in infancy produces some asthma-like symptoms and may be followed by a recurrent wheeze later in the childhood. It has been proposed that RSV infection stimulates type 2 cytokine responses, akin to those found in atopy and asthma. We obtained peripheral blood cells from RSV-infected infants (n=30) and healthy controls (n=10). After restimulating them in vitro, intracellular IL-4 and IFN-g were measured by flow cytometry. Cells from RSV-infected infants produced more IL-4 and less IFN-g than healthy controls. Interleukin-4 production was more frequent in CD8 than CD4 cells, and the bias towards IL-4 production was the greatest in infants with mild infections. Suprisingly, B-lymphocytes in peripheral blood produced IL-4 as well. Our conclusions are that RSV infection is associated with IL-4 production in peripheral T cells, and that peripheral blood in infants with severe disease may be depleted of cytokine-producing cells.
Sažetak: Toll-like receptors (TLRs) are a family of germ-line encoded proteins involved in host d... more Sažetak: Toll-like receptors (TLRs) are a family of germ-line encoded proteins involved in host defense by their ability to recognize conserved components of different microorganisms. According to current data, TLRs that recognize bacterial and fungal cell ...
Respiratory syncytial virus (RSV) causes acute respiratory tract illness across all ages while lo... more Respiratory syncytial virus (RSV) causes acute respiratory tract illness across all ages while lower respiratory tract infection (LRTI) is usually a result of primary infection in infants. Supportive care is the mainstay of therapy and includes oxygenotherapy, use of bronchodilators and corticosteroids, although no convincing data exist for their efficacy. Antimicrobial innate defense along with transferred maternal immunoglobulins play a significant role in infants while immune system undergoes conditional maturation. Beta- defensins are small antimicrobial peptides produced by epithelial cells and mononuclear leukocytes with wide pathogen specificity, displaying neutralization/inactivation capability. Even more, premature newborns show lower beta- defensin 1 production that might compromise early innate response. We determined levels of beta-defensin 1 and 2, IL- 1beta, TNF-alpha and IL-6 in BAL samples from infants with RSV-caused LRTI and infants with bacterial pneumonia in order to determine a profile of secreted antimicrobials. Infants admitted to ICU with confirmed RSV- infection (N=13) and infants with bacterial pneumonia (N=8) underwent to bronchoscopy. Infants with human metapneumo virus (HMPV) infection (N=3) were also included. High levels of both beta- defensins were detectable in majority of infants with higher levels in infants with acute RSV infection compared to infants with bacterial pneumonia and HMPV infection. Beta-defensin levels correlated with levels of pro-inflammatory TNF- alpha and IL- 1beta. Although, RSV and bacteria could both stimulate secretion of beta-defensins via pro- inflammatory cytokine loop, higher levels of beta- defensins 1 and 2 in acute RSV infection. Sustained high level of antimicrobial production by RSV challenged epithelial cells and infiltrated leukocytes could augment local inflammation and emphasize immunopathology seen in RSV infection.
Nije razjasnjeno znacenje P-glikoproteina (Pgp), biljega visestruke otpornosti na lijekove (engl.... more Nije razjasnjeno znacenje P-glikoproteina (Pgp), biljega visestruke otpornosti na lijekove (engl. multidrug resistance, MDR) u kronicnoj B-limfocitnoj leukemiji (B-CLL). Cilj istraživanja bio je odrediti da li MDR-status pri dijagnozi bolesti i tijekom lijecenja korelira s klinickim pokazateljima i ishodom lijecenja. Stoga smo određivali razinu Pgp-a u perifernoj krvi 42 bolesnika s B-CLL-om i 36 zdravih dobrovoljaca koristeci protocni citometar i monoklonska antitijela (mAt) na intracelularne (C-219) i ekstracelularne (MRK-16) epitope Pgp-a. Rezultati su izraženi kao omjer intenziteta fluorescencija mAt i izotipske kontrole (engl. ratio of mean of fluorescences, RMF). Pokazano je da su u zdravim ispitanicima kako intra tako i ekstracelularni biljezi Pgp-a znacajno (p<0.001) poviseni u starijih osoba. U nelijecenih bolesnika s B-CLL-om nađene su vise vrijednosti za RMF (medijani C-219=3.1, MRK-16=2, 9) u odnosu na lijecene (1, 9 odn. 2, 0). Ove vrijednosti nisu korelirale s klinickim pokazateljima (TTM ili RAI-status). Nadalje smo pratili bolesnike koji su primali terapiju s MDR-neovisnim lijekovima (klorambucil ili FAMP). U bolesnika koji su odgovorili na lijecenje (kompletna ili djelomicna remisija) pokazan je znacajan pad vrijednosti za RMF (C-219 sa 6, 8 na 2, 4, a za MRK-16 s 3, 0 na 1, 4 ). U bolesnika koji nisu odgovorili na terapiju vrijednosti Pgp-a su porasle ili se nisu mijenjale. Nasi rezultati upucuju da ekspresija Pgp-a korelira s terapijskim odgovorom cak i u lijekova ciji mehanizam djelovanja nije ovisan o Pgp-u.
Background: IaIp are group of serine proteases inhibitors. They are important in vivo modulators ... more Background: IaIp are group of serine proteases inhibitors. They are important in vivo modulators of endogenous proteases including trypsin, human leukocyte elastase, plasmin and cathepsin G. Release of endogenous proteases plays an important role in inflammation, sepsis, wound healing and metastasis. A significant decrease of plasma IaIp levels occurs in adult and newborn sepsis. Our previous animal studies using the polymicrobial sepsis rat (adult) model of cecal ligation and puncture demonstrated the beneficial effects of intravenous IaIp administration in improving morbidity and mortality. Objective: The aim of this study is to evaluate the effects of parenterally administered IaIp in an in vivo animal model of neonatal sepsis. Design/Methods: Sepsis was induced in 2-3 days old Fischer rats with a subcutaneous injection of E. Coli (BORT). A bacterial dose response curve of lethality was determined. 2 CFU/gram body weight was lethal at 40-50 hours after the infection. In the subsequent experiment, three day old animals (n=8) were injected with this dose of E. Coli and then randomized into two groups. The treatment group (n=4) received three intraperitoneal injections of 250 micrograms of purified human IaIp, at 6, 12 and 24 hours after the infection. The control group received equal amount of human albumin at the same time intervals. Results: All pups in the control group died within 40-44 hour after the infection. Animals in the treatment group survived up to 78 hours. Conclusions: These results suggest that IaIp offers a beneficial effect in septic newborn rats and warrant further investigation. Questions being pursued include: optimal dose vs. response to IaIp, optimal timing of IaIp administration, pharmacokinetics of IaIp, effectiveness of IaIp in other models of sepsis (E.g. Group B strep, LPS).
Respiratory syncytial virus (RSV) is the most common seasonal viral pathogen in children by secon... more Respiratory syncytial virus (RSV) is the most common seasonal viral pathogen in children by second year of life and in some cases it causes a severe respiratory disease. Frequent RSV reinfections and similarity of clinical findings with allergic asthma (airway hyper-responsiveness, wheezing) indicate development of mixed type- 1/type-2 immune response. Presence of IFN-γ/IL-4 in acute RSV infection could be confirmed by simultaneous production of type-1 associated (CXCL10) and type-2 associated (CCL17) chemokines, responsible for recruitment of activated CXCR3+ and CCR4+ T cells detected at higher percentages in the periphery of diseased infants indicating inadequate immune response. As CCR4 is not type-2 exclusive chemokine receptor, and could be found on different T cell types, addition of CRTh2 chemokine receptor analysis would contribute to more accurate type-2 immune response assessment. Moreover, most of conclusions regarding aberrant type of anti-RSV immune response in humans are extrapolated from peripheral blood analysis, and acquisition of bronchoalveolar lavage (BAL) samples of infected infants would provide unique insight into development of local immune response. BAL samples were obtained from infants hospitalized due to sever bronchiolitis or pneumonia caused by RSV (n=14) or other respiratory viruses (n=8) and bacterial pathogens (n=7) where bronchoscopy was clinically ascribed. Concentrations of chemokines CXCL10 and CCL17 were assessed using commercially available ELISA kits. Isolated cells were immunophenotyped for CD4 and CD8 T cell specific markers and specific chemokine receptors CXCR3, CCR4 and CRTh2. Cells were analysed on LSRII flow cytometer and data analysed using Statistica v7.0 software. BAL samples of all infected infants were found positive only for CXCL10, while CCL17 was undetectable regardless of pathogen. RSV infected infants had higher percentage CXCR3+ CD4 and CD8 T cells compared to other viral respiratory infections. High percentages of CCR4+ CD4 and CD8 T cells were also detected in RSV infected infants. Coexpression of CRTh2 chemokine receptor was detected on CCR4+ CD4 and CD8 T cells in RSV infected infants. The presence of type-2 immune response in lungs of RSV infected infants was confirmed in regards of higher expression of CCR4 and CRTh2 chemokine receptors on CD4 and CD8 T cells. Although CCL17 was not detected at the time of bronchoscopy, a high CXCL10 concentration implicates efficient tracking of antiviral and pro-inflammatory CXCR3+ T cell to the lungs. As CCL17 and CCR4 are, beside type-2 T cells, also involved in regulatory T cell (Treg) migration, undetectable CCL17 in RSV infected infants could explain more severe disease due to insufficient number of Treg cells in affected tissue.
Innate immunity is important part of immune system with ability to recognize and act (neutralize)... more Innate immunity is important part of immune system with ability to recognize and act (neutralize) upon pathogen challenge. Infants are particularly sensitive to different viral infections with emphasized detrimental effect of respiratory syncytial virus (RSV) induced recurrent infections in infancy related to inadequate anti-viral immunity post-infection. Toll-like receptors (TLRs) are part of the immune system able to recognize and activate immune system upon pathogen challenge. TLR3, 7, 8 recognize genomic viral RNA molecules initiating antiviral control mechanisms. Possible explanation for frequent RSV re-infections in infancy could be inadequate TLR3, 7, 8 engagement in innate immunity responses resulting in lower cytokines synthesis and dendritic cell maturation, due to lower TLRs expression in peripheral blood lymphocytes. In our study specific monoclonal antibodies against human TLR3, 7, 8 molecules were used to determine expression profile of peripheral blood leukocyte subpopulations from healthy and RSV-infected infants by flow cytometry. We have found that RSV-infected infants had lower percentages of TLR3-expressing monocytes, NK cells, T and B cells than healthy controls. Although expression of TLR7 in different leukocyte populations didn’ t statistically differ between healthy and infected infants, a tendency of lower percentages of TLR7 in monocytes, T and B cells was observed. The TLR8, cognate of TLR7 molecule, is expressed on smaller percentage of NK and T cells in RSV infected infants. The comparison of TLR7 and TLR8 expression between different age groups revealed lower expression in monocytes from peripheral blood of infants (diseased and healthy group) than in monocytes from healthy adults and newborns. In conclusion, lower expression of TLR3, 7, 8 stresses out inadequate early antiviral and innate immune mechanisms responsible for the RSV replication control and generating an effective immune response.
Toll-like receptors (TLRs) are part of the innate immune system able to recognize pathogen-associ... more Toll-like receptors (TLRs) are part of the innate immune system able to recognize pathogen-associated molecular patterns and activate immune system upon pathogen challenge. TLR7 and TLR8 recognize single-stranded viral RNA and initiate antiviral control mechanisms. A RNA virus particularly detrimental in infancy is respiratory syncytial virus (RSV). RSV can cause severe lower respiratory tract disease and recurrent infections related to inadequate development of anti-viral immunity. The reason for frequent RSV re-infections in infancy could be inadequate TLR7 and TLR8 engagement by viral RNA resulting in lower synthesis of anti-inflammatory mediators and antigen presentation, possibly due to a lower TLR expression in peripheral blood mononuclear cells (PBMC) of a still developing immune system. We determined TLR7 and TLR8 expression in PBMC subsets from infants with primary RSV infection and their healthy controls using multi-parameter flow cytometry. We found the expression of TLR7 and TLR8 in monocytes lower in infected infants than in healthy controls. Also, the comparison of TLR7 and TLR8 expression between different age groups revealed that they are expressed at lower level in monocytes from infants (both diseased and healthy) when compared to healthy adults and newborns. Unexpectedly, TLR8 in T cells and NK cells was detected in lower percentage in infected infants when compared with healthy controls. Difference in percentage of both cell subsets positive for TLR8 between healthy and infected infants emphasize possible role in mounting anti-RSV immunoreactions. In conclusion, difference in TLR7 and TLR8 expression stresses out their importance in early antiviral immune mechanisms responsible for the control of RSV replication and generation of an adaptive immune response.
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