Q-fever in Primorsko-goranska County from 1999 to 2005

Q-groznica je jedna od vodecih zoonoza u našoj zemlji koja u pojedinacnim slucajevima najcešce ostaje neprepoznata. U radu je analizirano kretanje Q-groznice tijekom šestogodišnjeg razdoblja od 1999. do 2005. godine u Primorsko-goranskoj županiji. Ukupno je prijavljeno 75 slucajeva Q-groznice, od toga je 58 (77%) muškaraca, najviše u dobnoj skupini od 21-30 godina – 18 (24%). Bolest se najcešce javljala u periodima kasne zime i ranog proljeca Hospitalizirano je svega 15 bolesnika. Smrtnih slucajeva nije bilo. Posebno je obradena epidemija Q-groznice koja je izbila 2004. godine u Novom Vindolskom i njegovoj okolici.Q-fever (Query fever) is one of the leading zoonosis in our country, which, in particular cases, usually remains undetectable. A six-year period in Primorsko-goranska County, from 1999 to 2005, has been analyzed in the study. A total of 75 cases concerning Q-fever have been reported, out of which 58 (77%) involved men, mostly between 21 and 30 years of age – 18 (24%). The disease usually appears in the late winter or early spring. There have been no cases having mortal consequences and only 15 patients have been hospitalized. A special emphasis and research has been given to the Q-fever epidemics that occurred in the year 2004 in Novi Vinodolski and its outskirts

EXPRESSION AND FUNCTION OF GRANULYSIN AND PERFORIN IN THE FIRST TRIMESTER PREGNANCY DECIDUA

Cilj istraživanja: Ispitali smo: (1) prisutnost granulizinskih oblika u decidualnim limfocitima i limfocitima periferne krvi, (2) stupanj kolokalizacije oblika granulizina s perforinom i biljegom degranulacije LAMP-1 nakon aktivacije stanica, (3) utjecaj molekula HLA-C i HLA-G, te IL-15, IL-2 i HSP70 na stupanj kolokalizacije, (4) utjecaj molekula HSP70 i PIBF na izražaj granulizina i perforina u decidualnim limfocitnim subpopulacijma, (5) mehanizam citotoksičnosti decidualnih limfocita (6) prisutnost perforina-2 na majčinoembrijskom spoju zdrave i patoloških trudnoća, (7) utjecaj interferona na izražaj gRNA za granulizin, perforin i perforin-2. Materijal i metode: Uzorke decidualnog tkiva i perifernu krv dobivali smo s Klinike za ginekologiju i porodništvo KBC-a Rijeka. Korištene metode su: imunofluorescencija, Western blot, test citotoksičnosti, RT-qPCR i imunohistokemija. Rezultati: Aktivacijom decidualnih limfocita dolazi do povišene skolokalizacije oblika granulizina od 9 kDa i perforina s LAMP-1. Molekula HLA-C u decidualnim limfocitima snižava kolokalizaciju oblika granulizina od 9 kDa s perforinom, dok molekula HLA-G potiče kolokalizaciju oblika od 15 kDa. IL-15 u decidualnim limfocitima smanjuje stupanj kolokalizacije oblika granulizina od 9 kDa s LAMP-1. Molekula HSP70 u decidualnim limfocitima potiče kolokalizaciju oba oblika granulizina s LAMP-1. Blokiranje protugranulizinskim i protuperforinskim protutijelima smanjuje nekrozu ciljnih stanica u testu citotoksičnosti. Perforin-2 je izražan u zdravoj trudnoći i patološkim trudnoćama, a IFN-γ povećava izražaj gRNA za perforin-2. Zaključak: Ovo istraživanje po prvi puta ukazuju na važnost kolokalizacije oblika granulizina i perforina kao čimbenika apoptotičkog djelovanja na majčino-embrijskom spoju.Aim of the study: We investigated the: (1) expression of granulysin forms in decidual and peripheral blood lymphocytes, (2) colocalization of granulysin forms with perforin and LAMP-1 (marker of degranulation) after cell activation, (3) influence of HLA-C and HLAG molecules, and IL-15, IL-2 and HSP70 on colocalization, (4) influence of HSP70 and PIBF molecules on granulysin and perforin expression in decidual lymphocytes subpopulations, (5) cytotoxicity mechanism of decidual lymphocytes, (6) expression of perforin-2 in healthy and pathological pregnancies, (7) influence of interferons on mRNA expression for granulysin, perforin and perforin-2. Materials and methods: Decidual tissue and peripheral blood were obtained from Clinic of Gynecology and Obstetrics, Clinical Hospital Centre of Rijeka. We have used immunofluorescence, Western blotting, cytotoxicity assay, RT-qPCR and immunohistology. Results: After activation, 9 kDa granulysin form and perforin colocalize more with LAMP1. In decidual lymphocytes HLA-C molecule decreased the colocalization of 9 kDa granulysin with perforin, while HLA-G molecule increased the colocalization of 15 kDa granulysin with perforin. IL-15 in decidual lymphocytes decreased the colocalization of 9 kDa granulysin form with LAMP-1. HSP70 increased the colocalization of both granulysin forms with LAMP-1. By using using anti-granulysin and anti-perforin antibodies we have decreased the target cell necrosis. Perforin-2 is expressed in both healthy and pathological pregnancies, while IFN-γ upregulates the mRNA for perforin-2. Conclusion: This investigation for the first time suggests the importance of granulysin form and perforin colocalization, as a factor of cytotoxic and cytolytic activity at the maternal-embryonic interface

EXPRESSION AND FUNCTION OF GRANULYSIN AND PERFORIN IN THE FIRST TRIMESTER PREGNANCY DECIDUA

Cilj istraživanja: Ispitali smo: (1) prisutnost granulizinskih oblika u decidualnim limfocitima i limfocitima periferne krvi, (2) stupanj kolokalizacije oblika granulizina s perforinom i biljegom degranulacije LAMP-1 nakon aktivacije stanica, (3) utjecaj molekula HLA-C i HLA-G, te IL-15, IL-2 i HSP70 na stupanj kolokalizacije, (4) utjecaj molekula HSP70 i PIBF na izražaj granulizina i perforina u decidualnim limfocitnim subpopulacijma, (5) mehanizam citotoksičnosti decidualnih limfocita (6) prisutnost perforina-2 na majčinoembrijskom spoju zdrave i patoloških trudnoća, (7) utjecaj interferona na izražaj gRNA za granulizin, perforin i perforin-2. Materijal i metode: Uzorke decidualnog tkiva i perifernu krv dobivali smo s Klinike za ginekologiju i porodništvo KBC-a Rijeka. Korištene metode su: imunofluorescencija, Western blot, test citotoksičnosti, RT-qPCR i imunohistokemija. Rezultati: Aktivacijom decidualnih limfocita dolazi do povišene skolokalizacije oblika granulizina od 9 kDa i perforina s LAMP-1. Molekula HLA-C u decidualnim limfocitima snižava kolokalizaciju oblika granulizina od 9 kDa s perforinom, dok molekula HLA-G potiče kolokalizaciju oblika od 15 kDa. IL-15 u decidualnim limfocitima smanjuje stupanj kolokalizacije oblika granulizina od 9 kDa s LAMP-1. Molekula HSP70 u decidualnim limfocitima potiče kolokalizaciju oba oblika granulizina s LAMP-1. Blokiranje protugranulizinskim i protuperforinskim protutijelima smanjuje nekrozu ciljnih stanica u testu citotoksičnosti. Perforin-2 je izražan u zdravoj trudnoći i patološkim trudnoćama, a IFN-γ povećava izražaj gRNA za perforin-2. Zaključak: Ovo istraživanje po prvi puta ukazuju na važnost kolokalizacije oblika granulizina i perforina kao čimbenika apoptotičkog djelovanja na majčino-embrijskom spoju.Aim of the study: We investigated the: (1) expression of granulysin forms in decidual and peripheral blood lymphocytes, (2) colocalization of granulysin forms with perforin and LAMP-1 (marker of degranulation) after cell activation, (3) influence of HLA-C and HLAG molecules, and IL-15, IL-2 and HSP70 on colocalization, (4) influence of HSP70 and PIBF molecules on granulysin and perforin expression in decidual lymphocytes subpopulations, (5) cytotoxicity mechanism of decidual lymphocytes, (6) expression of perforin-2 in healthy and pathological pregnancies, (7) influence of interferons on mRNA expression for granulysin, perforin and perforin-2. Materials and methods: Decidual tissue and peripheral blood were obtained from Clinic of Gynecology and Obstetrics, Clinical Hospital Centre of Rijeka. We have used immunofluorescence, Western blotting, cytotoxicity assay, RT-qPCR and immunohistology. Results: After activation, 9 kDa granulysin form and perforin colocalize more with LAMP1. In decidual lymphocytes HLA-C molecule decreased the colocalization of 9 kDa granulysin with perforin, while HLA-G molecule increased the colocalization of 15 kDa granulysin with perforin. IL-15 in decidual lymphocytes decreased the colocalization of 9 kDa granulysin form with LAMP-1. HSP70 increased the colocalization of both granulysin forms with LAMP-1. By using using anti-granulysin and anti-perforin antibodies we have decreased the target cell necrosis. Perforin-2 is expressed in both healthy and pathological pregnancies, while IFN-γ upregulates the mRNA for perforin-2. Conclusion: This investigation for the first time suggests the importance of granulysin form and perforin colocalization, as a factor of cytotoxic and cytolytic activity at the maternal-embryonic interface

EXPRESSION AND FUNCTION OF GRANULYSIN AND PERFORIN IN THE FIRST TRIMESTER PREGNANCY DECIDUA

Cilj istraživanja: Ispitali smo: (1) prisutnost granulizinskih oblika u decidualnim limfocitima i limfocitima periferne krvi, (2) stupanj kolokalizacije oblika granulizina s perforinom i biljegom degranulacije LAMP-1 nakon aktivacije stanica, (3) utjecaj molekula HLA-C i HLA-G, te IL-15, IL-2 i HSP70 na stupanj kolokalizacije, (4) utjecaj molekula HSP70 i PIBF na izražaj granulizina i perforina u decidualnim limfocitnim subpopulacijma, (5) mehanizam citotoksičnosti decidualnih limfocita (6) prisutnost perforina-2 na majčinoembrijskom spoju zdrave i patoloških trudnoća, (7) utjecaj interferona na izražaj gRNA za granulizin, perforin i perforin-2. Materijal i metode: Uzorke decidualnog tkiva i perifernu krv dobivali smo s Klinike za ginekologiju i porodništvo KBC-a Rijeka. Korištene metode su: imunofluorescencija, Western blot, test citotoksičnosti, RT-qPCR i imunohistokemija. Rezultati: Aktivacijom decidualnih limfocita dolazi do povišene skolokalizacije oblika granulizina od 9 kDa i perforina s LAMP-1. Molekula HLA-C u decidualnim limfocitima snižava kolokalizaciju oblika granulizina od 9 kDa s perforinom, dok molekula HLA-G potiče kolokalizaciju oblika od 15 kDa. IL-15 u decidualnim limfocitima smanjuje stupanj kolokalizacije oblika granulizina od 9 kDa s LAMP-1. Molekula HSP70 u decidualnim limfocitima potiče kolokalizaciju oba oblika granulizina s LAMP-1. Blokiranje protugranulizinskim i protuperforinskim protutijelima smanjuje nekrozu ciljnih stanica u testu citotoksičnosti. Perforin-2 je izražan u zdravoj trudnoći i patološkim trudnoćama, a IFN-γ povećava izražaj gRNA za perforin-2. Zaključak: Ovo istraživanje po prvi puta ukazuju na važnost kolokalizacije oblika granulizina i perforina kao čimbenika apoptotičkog djelovanja na majčino-embrijskom spoju.Aim of the study: We investigated the: (1) expression of granulysin forms in decidual and peripheral blood lymphocytes, (2) colocalization of granulysin forms with perforin and LAMP-1 (marker of degranulation) after cell activation, (3) influence of HLA-C and HLAG molecules, and IL-15, IL-2 and HSP70 on colocalization, (4) influence of HSP70 and PIBF molecules on granulysin and perforin expression in decidual lymphocytes subpopulations, (5) cytotoxicity mechanism of decidual lymphocytes, (6) expression of perforin-2 in healthy and pathological pregnancies, (7) influence of interferons on mRNA expression for granulysin, perforin and perforin-2. Materials and methods: Decidual tissue and peripheral blood were obtained from Clinic of Gynecology and Obstetrics, Clinical Hospital Centre of Rijeka. We have used immunofluorescence, Western blotting, cytotoxicity assay, RT-qPCR and immunohistology. Results: After activation, 9 kDa granulysin form and perforin colocalize more with LAMP1. In decidual lymphocytes HLA-C molecule decreased the colocalization of 9 kDa granulysin with perforin, while HLA-G molecule increased the colocalization of 15 kDa granulysin with perforin. IL-15 in decidual lymphocytes decreased the colocalization of 9 kDa granulysin form with LAMP-1. HSP70 increased the colocalization of both granulysin forms with LAMP-1. By using using anti-granulysin and anti-perforin antibodies we have decreased the target cell necrosis. Perforin-2 is expressed in both healthy and pathological pregnancies, while IFN-γ upregulates the mRNA for perforin-2. Conclusion: This investigation for the first time suggests the importance of granulysin form and perforin colocalization, as a factor of cytotoxic and cytolytic activity at the maternal-embryonic interface

Colocalization of Granulysin Protein Forms with Perforin and LAMP-1 in Decidual Lymphocytes During Early Pregnancy

PROBLEM: Granulysin (GNLY) occurs in two forms, which have molecular weights of 9 and 15 kDa. We analyzed the cytotoxic potential of decidual lymphocytes (DLs) and peripheral blood lymphocytes (PBLs) based on the forms of GNLY that colocalizes with perforin (PER) and LAMP-1 following activation. METHODS: The forms of GNLY were detected by using confocal microscopy. We investigated the colocalization with PER and LAMP-1 in freshly isolated and activated DLs and PBLs. RESULTS: Activation of DLs and PBLs by K- 562 cells increased the colocalization of 9 kDa GNLY with PER and LAMP- 1. K-562 cells transfected with HLA-C decreased 9 kDa GNLY colocalization with PER in DLs only. IL-15 in DLs decreased 9 kDa GNLY and LAMP-1 colocalization, but increased both 15 kDa GNLY and LAMP-1, and PER and LAMP-1 colocalization. CONCLUSION: Activated DLs and PBLs show greater cytotoxic potential based on increased colocalization of 9 kDa GNLY and PER. HLA-C and IL-15 affect DLs, indicating their role in maintaining the pregnancy tolerance

Colocalization of Granulysin Protein Forms with Perforin and LAMP-1 in Decidual Lymphocytes During Early Pregnancy

PROBLEM: Granulysin (GNLY) occurs in two forms, which have molecular weights of 9 and 15 kDa. We analyzed the cytotoxic potential of decidual lymphocytes (DLs) and peripheral blood lymphocytes (PBLs) based on the forms of GNLY that colocalizes with perforin (PER) and LAMP-1 following activation. METHODS: The forms of GNLY were detected by using confocal microscopy. We investigated the colocalization with PER and LAMP-1 in freshly isolated and activated DLs and PBLs. RESULTS: Activation of DLs and PBLs by K- 562 cells increased the colocalization of 9 kDa GNLY with PER and LAMP- 1. K-562 cells transfected with HLA-C decreased 9 kDa GNLY colocalization with PER in DLs only. IL-15 in DLs decreased 9 kDa GNLY and LAMP-1 colocalization, but increased both 15 kDa GNLY and LAMP-1, and PER and LAMP-1 colocalization. CONCLUSION: Activated DLs and PBLs show greater cytotoxic potential based on increased colocalization of 9 kDa GNLY and PER. HLA-C and IL-15 affect DLs, indicating their role in maintaining the pregnancy tolerance

Assessing whether progesterone-matured dendritic cells are responsible for retention of fertilization products in missed abortion

We hypothesize that progesterone causes tolerogenic maturation of myeloid dendritic cells (DCs) in human decidua of threatened miscarriage or missed abortion characterized by a distinct phenotype and cytokine production, including reduction of the main NK cell proliferation and cytotoxic factor interleukin (IL)-15. During DC/NK cell interaction, progesterone-shaped DCs cannot efficiently multiply or equip NK cells with the cytotoxic mediators peforin and granulysin, which might harm trophoblasts and induce abortion. We propose that the presence, and maturation stage of decidual myeloid DCs be investigated using semi-quantitative immunohistological analyses and/or double-color immuno-fluorescent labeling of DC lineage and activation markers. The spatial arrangement of granulysin+ cells, NKp46+ NK cells, DCs, and trophoblasts might provide information about their mutual interactions in vivo. Multiple flow cytometry analyses of NK-receptors would provide insight into NK cell activation status. NK cell activation status could be also assessed by cytotoxicity assays against trophoblast cell lines, or isolated cognate extra-villous trophoblast cells. A correlation between decidual progesterone concentration or IL-15 expression, and the degree of DC maturation or the frequency of granulysin+ cells, might help to elucidate the mechanism of abortion retention in utero

Granulysin-mediated apoptosis of trophoblasts in blighted ovum and missed abortion

Granulysin (GNLY) is a cytotoxic molecule mostly present in decidual natural killer (NK) cells. Blighted ovum (BO) and missed abortion (MA) represent the early pathological pregnancies with hindered development of the embryoblast or a dead embryo. We investigated the GNLY-mediated apoptotic mechanism potentially responsible for delayed termination of pregnancy. METHOD OF STUDY: We performed immunohistological and immunofluorescence labeling of decidual tissues (GNLY, Apaf-1, NF-κB). NKG2A expression was analyzed by flow cytometry and GNLY mRNA by RT- qPCR. RESULTS: The GNLY labeling intensity (H score) was lower in the nuclei of trophoblast cells in BO and MA. GNLY gene levels were inversely detected in BO and MA. A decreased decidual NK cell percentage was found in MA. NK cells from pathological pregnancies expressed lower NKG2A levels. The highest frequency of Apaf-1 was found in trophoblast cells of MA. NF- kB was highly expressed in decidual cells of BO. CONCLUSION: The reduced activation of GNLY- mediated killing might be implicated in the slower rejection of trophoblast cells in BO and MA. A decreased authentic decidual NK cell number could be responsible for low cytotoxicity against trophoblast cells in MA. In BO, trophoblast cells have a higher survival potential due to increased NF-kB expressio

Potential role of heat-shock protein 70 and interleukin-15 in the pathogenesis of threatened spontaneous abortions

The role of HSP70 and both its constitutive (Hsc) and inducible (Hsp) forms in the pathogenesis of threatened spontaneous abortions was investigated. Immunohistology and/or immunofluorescence was used to analyze paraffin- embedded tissue sections, and reverse transcriptase-quantitative polymerase chain reaction and flow cytometry were used for analyses of decidual mononuclear cells (DMCs) and confocal microscopy for the detection of perforin, granulysin, and lysosome-associated membrane protein-1 (LAMP-1) in decidual lymphocytes (DLs). The percentage of single Hsp70+, Hsc70+, and IL-15+ cells and mRNA levels of HSP70, CD91, and TLR4 were lower in the decidua basalis in cases of threatened miscarriages compared to that in cases of normal pregnancy. In a suspension of normal DMCs, IL-15 significantly decreased the HSP70 members and TLR4 in dendritic cells, T cells, and NK cells while increasing CD91 in NK cells alone. Downregulation of Hsc70, Hsp70, and IL-15 expression at gene and/or protein levels might support the retention of fertilization products in cases of missed abortion and blighted ovum

Neuroimmune characterization of optineurin insufficiency mouse model during ageing

Optineurin is a multifunctional polyubiquitin-binding protein implicated in infammatory signalling. Optineurin mutations are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), neurodegenerative diseases characterised by neuronal loss, neuroinfammation, and peripheral immune disbalance. However, the pathogenic role of optineurin mutations is unclear. We previously observed no phenotype in the unmanipulated young optineurin insufciency mice (Optn470T), designed to mimic ALS/FTD-linked truncations defcient in polyubiquitin binding. The purpose of this study was to investigate whether ageing would trigger neurodegeneration. We performed a neurological, neuropathological, and immunological characterization of ageing wild-type (WT) and Optn470T mice. No motor or cognitive diferences were detected between the genotypes. Neuropathological analyses demonstrated signs of ageing including lipofuscin accumulation and microglial activation in WT mice. However, this was not worsened in Optn470T mice, and they did not exhibit TAR DNA-binding protein 43 (TDP-43) aggregation or neuronal loss. Spleen immunophenotyping uncovered T cell immunosenescence at two years but without notable diferences between the WT and Optn470T mice. Conventional dendritic cells (cDC) and macrophages exhibited increased expression of activation markers in two-year-old Optn470T males but not females, although the numbers of innate immune cells were similar between genotypes. Altogether, a combination of optineurin insufciency and ageing did not induce ALS/FTD-like immune imbalance and neuropathology in mice