Uloga metalotionina u karcinomu dojke

Metallothioneins (MTs) are a family of small cysteine-rich proteins involved in many physiological and pathological processes. Genes that encode the four isoforms of metallothioneins (MT1-M4) are located on chromosome 16q13. Strucutures of these four isoforms allow metallothioneins their various biological functions. Many studies have shown that MT plays an important role in carcinogenesis, tumour growth, its progression from local to metastatic disease and may contribute to resistence to chemotherapy and radiotherapy. Due to the fact that breast cancer is one of the leading causes of death in women worldwide it is important to better understand the biology of breast cancer. So, findings of MT could evenutally help as a prognostic tool and could lead to a possible new specific anti-cancer treatment.Metalotioneini su skupina proteina bogatih cisteinima koji su uključeni u mnoge fiziološke i patološke procese. Geni koji kodiraju četiri izoforme metalotioneina locirani su na lokusu 16q13. Strukture ovih četiriju izoformi omogućuju metalotioneinima njihove razne biološke funkcije. Mnoge su studije pokazale da metalnotionein ima važnu ulogu u karcinogenezi, rastu tumora, njegovoj progresiji od lokalne prema metastatskoj bolesti te je povezan sa razvojem rezistencije na kemoterapiju i radioterapiju. Rak dojke jedan je od vodećih uzroka smrti u svijetu i važno je bolje razumijeti ulogu metalotioneina u različitim podtipovima karcinoma dojke. Ovakvi podaci mogli bi pomoći kao prognostički alat i voditi pronalasku novog specifičnog liječenja karcinoma

Synthetic vs natural scaffolds for human limbal stem cells

Aim To investigate the impact of synthetic electrospun polyurethane (PU) and polycaprolactone (PCL) nanoscaffolds, before and after hydrolytic surface modification, on viability and differentiation of cultured human eye epithelial cells, in comparison with natural scaffolds: fibrin and human amniotic membrane. Methods Human placenta was taken at elective cesarean delivery. Fibrin scaffolds were prepared from commercial fibrin glue kits. Nanoscaffolds were fabricated by electrospinning. Limbal cells were isolated from surpluses of human cadaveric cornea and seeded on feeder 3T3 cells. The scaffolds used for viability testing and immunofluorescence analysis were amniotic membrane, fibrin, PU, and PCL nanoscaffolds, with or without prior NaOH treatment. Results Scanning electron microscope photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. There was a significant difference in viability performance between cells with highest viability cultured on tissue culture plastic and cells cultured on all other scaffolds. On the other hand, electrospun PU, PCL, and electrospun PCL treated with NaOH had more than 80% of limbal cells positive for stem cell marker p63 compared to only 27%of p63 positive cells on fibrin. Conclusion Natural scaffolds, fibrin and amniotic membrane, showed better cell viability than electrospun scaffolds. On the contrary, high percentages of p63 positive cells obtained on these scaffolds still makes them good candidates for efficient delivery systems for therapeutic purpose

THE INFLUENCE OF HEAT SHOCK PROTEIN 70 AND GLYCOPROTEIN 96 ON MATURATION PROGRAM OF DECIDUAL CD1a+ DENDRITIC CELLS IN THE FIRST TRIMESTER OF PREGNANCY

Cilj istraživanja: Bjelančevine toplinskog šoka su visoko očuvane i ubikvitarne molekule prisutne unutar stanice na majčino-fetalnom spoju. Tijekom implantacije dolazi do opsežne tkivne pregradnje, pri čemu se u izvanstanični prostor mogu otpustiti bjelančevine toplinskog šoka. Snažno imunogeno djelovanje ovih bjelančevina može postati okidač za aktivaciju pro-upalnog imunološkog odgovora te prijetnja održavanju trudnoće. Cilj ovog rada bio je utvrditi obrazac izražaja bjelančevina toplinskog šoka 70 i gp96 (od engl. glycoprotein 96) te njihovih receptora CD91 i TLR4 (od engl. Toll Like Receptor 4) na mjestu prodora trofoblasta u decidualno tkivo prvog tromjesečja normalne i patoloških trudnoća (anembrijska trudnoća i zadržani pobačaj) te tkiva posteljice na terminskoj trudnoći. Nadalje, ispitati mehanizme nadziranja izražaja bjelančevina toplinskog šoka (HSP70 i gp96) i njihovih receptora CD91 i TLR4 pod utjecajem PIBF-a (od engl. Progesterone Induced Blocking Factor) i pro-upalnih citokina (IL-15 i IL-2). Također, proučiti utjecaj HSP70 i gp96 na fenotip i funkciju decidualnih CD1a+ dendritičkih stanica in vitro. Materijali i metode: Raspodjela Hsc70, Hsp70, gp96, CD91 i TLR4 u decidualnom tkivu prvog tromjesečja normalne i patoloških trudnoća te tkiva posteljice na terminskoj trudnoći uklopljenih u parafin ispitana je metodama imunohistologije i imunoflorescencije. Enzimatskom razgradnjom tkiva i centrifugiranjem na gradijentu gustoće izdvojene su decidualne mononuklearne stanice (DMS) i analiziran je utjecaj rastućih koncentracija PIBF-a, IL-15 i IL-2 na izražaj HSP70, gp96, CD91 i TLR4 protočnom citometrijom i RT-qPCR metodom. Vezanje HSP70 i gp96 za receptore CD91 i TLR4 na CD1a+ DS dokazano je testom specifičnog vezanja. Ispitan je utjecaj HSP70 i gp96 na fenotip, stupanj sazrijevanja, unutarstanično izražavanje citokina i kemokina u decidualnim CD1a+ DS protočnom citometrijom nakon 18-sati stimulacije in vitro. Rezultati: Hsc70, Hsp70, gp96, CD91 i TLR4 prisutni su u uzorcima decidualnog tkiva prvog tromjesečja zdrave i patoloških trudnoća te tkiva posteljice na terminskoj trudnoći i to posebno u žlijezdanom epitelu, endotelu kapilara, stanicama raspršenim u stromi decidue i stanicama trofoblasta. Pod utjecajem PIBF-a dolazi do smanjenja izražaja HSP70, gp96, CD91 i TLR4 na razini gena i bjelančevina u suspenziji DMS. Citokini, IL-2 i IL-15 smanjuju izražaj gRNK za HSP70, CD91 i TLR4, dok povećavaju izražaj gRNK za gp96. IL-2 također, smanjuje udio CD91+ i TLR4+ stanica u različitim staničnim subpopulacijama DMS-a. IL-15 povećava udio CD91+ stanica NK, ali smanjuje udio TLR4+ stanica NK, limfocita T, nezrelih i zrelih DS. HSP70 i gp96 specifično se vežu za CD91 i TLR4 na decidualnim CD1a+ DS. Pod utjecajem HSP70 i gp96 nezrele CD1a+ DS povećavaju izražaj CD83, HLA-DR, ko-stimulacijskih molekula (CD80 i CD86) te kemokina (CCL3 i CCL22) i citokina (INF- i IL-15). Zaključak: Bjelančevine toplinskog šoka (HSP70, gp96) i njihovi receptori (CD91 i TLR4) su prisutni na majčino-fetalnom spoju tijekom prvog tromjesečja zdrave i patoloških trudnoća te u tkivu posteljice na terminskoj trudnoći, što predstavlja molekulsku osnovu za njihovo međudjelovanje. Intenzitet izraženosti ovih molekula mogla bi biti posljedica složenih međuodnosa PIBF-a, IL-15 i IL-2. U pokusima in vitro, HSP70 i gp96 kao prirodni ligandi za CD91 i TLR4 potiču sazrijevanje i aktivaciju CD1a+ DS sa stvaranjem pretežno pro-upalnih medijatora, što može podržati štetan imunološki odgovor i nepovoljan ishod trudnoćeObjectives: Heat shock proteins are highly conserved and ubiquitous molecules present in the cells at the maternal-fetal interface. During the extensive tissue remodelling which is followed by the release of HSPs into the extracellular space. Strongly immunogenic activity of these proteins might bias the immune response towards the Th1 pattern and could cause undesirable pregnancy termination. The aim of the study was to evaluate the expression of HSP70 family members (Hsc70 and Hsp70), glycoprotein 96 (gp96) and their receptors CD91 and Toll like receptor 4 (TLR4) was investigated at maternal-fetal interface of the first trimester normal and pathological pregnancies (blighted ovum and missed abortion) and placental tissue at term. The mecchanisam of regulation of HSP70, gp96, CD91 and TLR4 expression was analysed by increasing concentration of Progesterone Induced Blocking Factor (PIBF), interleukin (IL)-15 and IL-2. The influence of HSP70 and gp96 on phenotype and maturation process of decidual CD1a+ dendritic cells (DC) was evaluated in vitro. Material and methods: Immunohistological and immunofluorescent labelling were performed on paraffin-embedded decidual sections of normal and pathological first trimester pregnancies (blighted ovum and missed abortion) and term placenta to detect gp96, Hsc70, Hsp70, CD91 and TLR4. The samples of normal first trimester pregnancy decidua were enzymatically digested and gradient density centrifuged to obtain the suspension of decidual mononuclear cells (DMC). DMC were stimulated with increasing concentration of PIBF, IL-15 and IL-2 and the expression of HSP70, gp96, CD91 and TLR4 was analysed by flow cytometry and RT-qPCR. The specific binding of HSP70 and gp96 was asset for receptors CD91 and TLR4 on CD1a+ DC. Flow cytometry was used to evaluate the influences of HSP70 and gp96 on the phenotype, the maturation process and the intracellular expression of cytokines and chemokines in decidual CD1a+ DC. Results: Hsc70, Hsp70, gp96, CD91 and TLR4 are present in decidual tissue of normal and pathological pregnancies first trimester (blighted ovum and missed abortion) and term placenta and they were found in glandular cells, endothelial cells, cells randomly distributed in stroma and trophoblast cells. PIBF down-regulated HSP70, gp96, CD91 and TLR4 at gene and proteins levels in DMCs. Cytokines, IL-2 and IL-15 decreased mRNA for HSP70, CD91 and TLR4, while increased mRNA for gp96. IL-2 also decreased the frequency of CD91+ and TLR4+ cells in different subpopulations of DMC. IL-15 treatment increased the frequency of CD91+ NK cells, but decreased the frequency of TLR4 expressing NK cells, T cells, immature and mature DC. HSP70 and gp96 efficiently binds CD91 and TLR4 receptors on decidual CD1a+ DC. In the presence of HSP70 and gp96 immature CD1a+ DC assume a more mature phenotype due to increased expression of CD83, HLA-DR, co-stimulatory CD80 and CD86 molecules, chemokines (CCL22 and CCL3) and cytokines (IFN-γ and IL-15). Conclusion: The presence of heat shock proteins (HSP70 and gp96) and their receptors CD91 and TLR4 at the maternal-fetal interface during normal and pathological first trimester pregnancies (blighted ovum and missed abortion) and term placenta provides a molecular basis for their interaction. The expression intensity of these molecules could be influenced by the interplay among PIBF, IL-15 and IL-2. HSP70 and gp96, as natural ligands for CD91 and TLR4 receptors induce maturation process of CD1a+ DC toward the expression of predominantly pro-inflammatory mediators, which could support a harmful immune response and adverse pregnancy outcome

THE INFLUENCE OF HEAT SHOCK PROTEIN 70 AND GLYCOPROTEIN 96 ON MATURATION PROGRAM OF DECIDUAL CD1a+ DENDRITIC CELLS IN THE FIRST TRIMESTER OF PREGNANCY

Cilj istraživanja: Bjelančevine toplinskog šoka su visoko očuvane i ubikvitarne molekule prisutne unutar stanice na majčino-fetalnom spoju. Tijekom implantacije dolazi do opsežne tkivne pregradnje, pri čemu se u izvanstanični prostor mogu otpustiti bjelančevine toplinskog šoka. Snažno imunogeno djelovanje ovih bjelančevina može postati okidač za aktivaciju pro-upalnog imunološkog odgovora te prijetnja održavanju trudnoće. Cilj ovog rada bio je utvrditi obrazac izražaja bjelančevina toplinskog šoka 70 i gp96 (od engl. glycoprotein 96) te njihovih receptora CD91 i TLR4 (od engl. Toll Like Receptor 4) na mjestu prodora trofoblasta u decidualno tkivo prvog tromjesečja normalne i patoloških trudnoća (anembrijska trudnoća i zadržani pobačaj) te tkiva posteljice na terminskoj trudnoći. Nadalje, ispitati mehanizme nadziranja izražaja bjelančevina toplinskog šoka (HSP70 i gp96) i njihovih receptora CD91 i TLR4 pod utjecajem PIBF-a (od engl. Progesterone Induced Blocking Factor) i pro-upalnih citokina (IL-15 i IL-2). Također, proučiti utjecaj HSP70 i gp96 na fenotip i funkciju decidualnih CD1a+ dendritičkih stanica in vitro. Materijali i metode: Raspodjela Hsc70, Hsp70, gp96, CD91 i TLR4 u decidualnom tkivu prvog tromjesečja normalne i patoloških trudnoća te tkiva posteljice na terminskoj trudnoći uklopljenih u parafin ispitana je metodama imunohistologije i imunoflorescencije. Enzimatskom razgradnjom tkiva i centrifugiranjem na gradijentu gustoće izdvojene su decidualne mononuklearne stanice (DMS) i analiziran je utjecaj rastućih koncentracija PIBF-a, IL-15 i IL-2 na izražaj HSP70, gp96, CD91 i TLR4 protočnom citometrijom i RT-qPCR metodom. Vezanje HSP70 i gp96 za receptore CD91 i TLR4 na CD1a+ DS dokazano je testom specifičnog vezanja. Ispitan je utjecaj HSP70 i gp96 na fenotip, stupanj sazrijevanja, unutarstanično izražavanje citokina i kemokina u decidualnim CD1a+ DS protočnom citometrijom nakon 18-sati stimulacije in vitro. Rezultati: Hsc70, Hsp70, gp96, CD91 i TLR4 prisutni su u uzorcima decidualnog tkiva prvog tromjesečja zdrave i patoloških trudnoća te tkiva posteljice na terminskoj trudnoći i to posebno u žlijezdanom epitelu, endotelu kapilara, stanicama raspršenim u stromi decidue i stanicama trofoblasta. Pod utjecajem PIBF-a dolazi do smanjenja izražaja HSP70, gp96, CD91 i TLR4 na razini gena i bjelančevina u suspenziji DMS. Citokini, IL-2 i IL-15 smanjuju izražaj gRNK za HSP70, CD91 i TLR4, dok povećavaju izražaj gRNK za gp96. IL-2 također, smanjuje udio CD91+ i TLR4+ stanica u različitim staničnim subpopulacijama DMS-a. IL-15 povećava udio CD91+ stanica NK, ali smanjuje udio TLR4+ stanica NK, limfocita T, nezrelih i zrelih DS. HSP70 i gp96 specifično se vežu za CD91 i TLR4 na decidualnim CD1a+ DS. Pod utjecajem HSP70 i gp96 nezrele CD1a+ DS povećavaju izražaj CD83, HLA-DR, ko-stimulacijskih molekula (CD80 i CD86) te kemokina (CCL3 i CCL22) i citokina (INF- i IL-15). Zaključak: Bjelančevine toplinskog šoka (HSP70, gp96) i njihovi receptori (CD91 i TLR4) su prisutni na majčino-fetalnom spoju tijekom prvog tromjesečja zdrave i patoloških trudnoća te u tkivu posteljice na terminskoj trudnoći, što predstavlja molekulsku osnovu za njihovo međudjelovanje. Intenzitet izraženosti ovih molekula mogla bi biti posljedica složenih međuodnosa PIBF-a, IL-15 i IL-2. U pokusima in vitro, HSP70 i gp96 kao prirodni ligandi za CD91 i TLR4 potiču sazrijevanje i aktivaciju CD1a+ DS sa stvaranjem pretežno pro-upalnih medijatora, što može podržati štetan imunološki odgovor i nepovoljan ishod trudnoćeObjectives: Heat shock proteins are highly conserved and ubiquitous molecules present in the cells at the maternal-fetal interface. During the extensive tissue remodelling which is followed by the release of HSPs into the extracellular space. Strongly immunogenic activity of these proteins might bias the immune response towards the Th1 pattern and could cause undesirable pregnancy termination. The aim of the study was to evaluate the expression of HSP70 family members (Hsc70 and Hsp70), glycoprotein 96 (gp96) and their receptors CD91 and Toll like receptor 4 (TLR4) was investigated at maternal-fetal interface of the first trimester normal and pathological pregnancies (blighted ovum and missed abortion) and placental tissue at term. The mecchanisam of regulation of HSP70, gp96, CD91 and TLR4 expression was analysed by increasing concentration of Progesterone Induced Blocking Factor (PIBF), interleukin (IL)-15 and IL-2. The influence of HSP70 and gp96 on phenotype and maturation process of decidual CD1a+ dendritic cells (DC) was evaluated in vitro. Material and methods: Immunohistological and immunofluorescent labelling were performed on paraffin-embedded decidual sections of normal and pathological first trimester pregnancies (blighted ovum and missed abortion) and term placenta to detect gp96, Hsc70, Hsp70, CD91 and TLR4. The samples of normal first trimester pregnancy decidua were enzymatically digested and gradient density centrifuged to obtain the suspension of decidual mononuclear cells (DMC). DMC were stimulated with increasing concentration of PIBF, IL-15 and IL-2 and the expression of HSP70, gp96, CD91 and TLR4 was analysed by flow cytometry and RT-qPCR. The specific binding of HSP70 and gp96 was asset for receptors CD91 and TLR4 on CD1a+ DC. Flow cytometry was used to evaluate the influences of HSP70 and gp96 on the phenotype, the maturation process and the intracellular expression of cytokines and chemokines in decidual CD1a+ DC. Results: Hsc70, Hsp70, gp96, CD91 and TLR4 are present in decidual tissue of normal and pathological pregnancies first trimester (blighted ovum and missed abortion) and term placenta and they were found in glandular cells, endothelial cells, cells randomly distributed in stroma and trophoblast cells. PIBF down-regulated HSP70, gp96, CD91 and TLR4 at gene and proteins levels in DMCs. Cytokines, IL-2 and IL-15 decreased mRNA for HSP70, CD91 and TLR4, while increased mRNA for gp96. IL-2 also decreased the frequency of CD91+ and TLR4+ cells in different subpopulations of DMC. IL-15 treatment increased the frequency of CD91+ NK cells, but decreased the frequency of TLR4 expressing NK cells, T cells, immature and mature DC. HSP70 and gp96 efficiently binds CD91 and TLR4 receptors on decidual CD1a+ DC. In the presence of HSP70 and gp96 immature CD1a+ DC assume a more mature phenotype due to increased expression of CD83, HLA-DR, co-stimulatory CD80 and CD86 molecules, chemokines (CCL22 and CCL3) and cytokines (IFN-γ and IL-15). Conclusion: The presence of heat shock proteins (HSP70 and gp96) and their receptors CD91 and TLR4 at the maternal-fetal interface during normal and pathological first trimester pregnancies (blighted ovum and missed abortion) and term placenta provides a molecular basis for their interaction. The expression intensity of these molecules could be influenced by the interplay among PIBF, IL-15 and IL-2. HSP70 and gp96, as natural ligands for CD91 and TLR4 receptors induce maturation process of CD1a+ DC toward the expression of predominantly pro-inflammatory mediators, which could support a harmful immune response and adverse pregnancy outcome

THE INFLUENCE OF HEAT SHOCK PROTEIN 70 AND GLYCOPROTEIN 96 ON MATURATION PROGRAM OF DECIDUAL CD1a+ DENDRITIC CELLS IN THE FIRST TRIMESTER OF PREGNANCY

Cilj istraživanja: Bjelančevine toplinskog šoka su visoko očuvane i ubikvitarne molekule prisutne unutar stanice na majčino-fetalnom spoju. Tijekom implantacije dolazi do opsežne tkivne pregradnje, pri čemu se u izvanstanični prostor mogu otpustiti bjelančevine toplinskog šoka. Snažno imunogeno djelovanje ovih bjelančevina može postati okidač za aktivaciju pro-upalnog imunološkog odgovora te prijetnja održavanju trudnoće. Cilj ovog rada bio je utvrditi obrazac izražaja bjelančevina toplinskog šoka 70 i gp96 (od engl. glycoprotein 96) te njihovih receptora CD91 i TLR4 (od engl. Toll Like Receptor 4) na mjestu prodora trofoblasta u decidualno tkivo prvog tromjesečja normalne i patoloških trudnoća (anembrijska trudnoća i zadržani pobačaj) te tkiva posteljice na terminskoj trudnoći. Nadalje, ispitati mehanizme nadziranja izražaja bjelančevina toplinskog šoka (HSP70 i gp96) i njihovih receptora CD91 i TLR4 pod utjecajem PIBF-a (od engl. Progesterone Induced Blocking Factor) i pro-upalnih citokina (IL-15 i IL-2). Također, proučiti utjecaj HSP70 i gp96 na fenotip i funkciju decidualnih CD1a+ dendritičkih stanica in vitro. Materijali i metode: Raspodjela Hsc70, Hsp70, gp96, CD91 i TLR4 u decidualnom tkivu prvog tromjesečja normalne i patoloških trudnoća te tkiva posteljice na terminskoj trudnoći uklopljenih u parafin ispitana je metodama imunohistologije i imunoflorescencije. Enzimatskom razgradnjom tkiva i centrifugiranjem na gradijentu gustoće izdvojene su decidualne mononuklearne stanice (DMS) i analiziran je utjecaj rastućih koncentracija PIBF-a, IL-15 i IL-2 na izražaj HSP70, gp96, CD91 i TLR4 protočnom citometrijom i RT-qPCR metodom. Vezanje HSP70 i gp96 za receptore CD91 i TLR4 na CD1a+ DS dokazano je testom specifičnog vezanja. Ispitan je utjecaj HSP70 i gp96 na fenotip, stupanj sazrijevanja, unutarstanično izražavanje citokina i kemokina u decidualnim CD1a+ DS protočnom citometrijom nakon 18-sati stimulacije in vitro. Rezultati: Hsc70, Hsp70, gp96, CD91 i TLR4 prisutni su u uzorcima decidualnog tkiva prvog tromjesečja zdrave i patoloških trudnoća te tkiva posteljice na terminskoj trudnoći i to posebno u žlijezdanom epitelu, endotelu kapilara, stanicama raspršenim u stromi decidue i stanicama trofoblasta. Pod utjecajem PIBF-a dolazi do smanjenja izražaja HSP70, gp96, CD91 i TLR4 na razini gena i bjelančevina u suspenziji DMS. Citokini, IL-2 i IL-15 smanjuju izražaj gRNK za HSP70, CD91 i TLR4, dok povećavaju izražaj gRNK za gp96. IL-2 također, smanjuje udio CD91+ i TLR4+ stanica u različitim staničnim subpopulacijama DMS-a. IL-15 povećava udio CD91+ stanica NK, ali smanjuje udio TLR4+ stanica NK, limfocita T, nezrelih i zrelih DS. HSP70 i gp96 specifično se vežu za CD91 i TLR4 na decidualnim CD1a+ DS. Pod utjecajem HSP70 i gp96 nezrele CD1a+ DS povećavaju izražaj CD83, HLA-DR, ko-stimulacijskih molekula (CD80 i CD86) te kemokina (CCL3 i CCL22) i citokina (INF- i IL-15). Zaključak: Bjelančevine toplinskog šoka (HSP70, gp96) i njihovi receptori (CD91 i TLR4) su prisutni na majčino-fetalnom spoju tijekom prvog tromjesečja zdrave i patoloških trudnoća te u tkivu posteljice na terminskoj trudnoći, što predstavlja molekulsku osnovu za njihovo međudjelovanje. Intenzitet izraženosti ovih molekula mogla bi biti posljedica složenih međuodnosa PIBF-a, IL-15 i IL-2. U pokusima in vitro, HSP70 i gp96 kao prirodni ligandi za CD91 i TLR4 potiču sazrijevanje i aktivaciju CD1a+ DS sa stvaranjem pretežno pro-upalnih medijatora, što može podržati štetan imunološki odgovor i nepovoljan ishod trudnoćeObjectives: Heat shock proteins are highly conserved and ubiquitous molecules present in the cells at the maternal-fetal interface. During the extensive tissue remodelling which is followed by the release of HSPs into the extracellular space. Strongly immunogenic activity of these proteins might bias the immune response towards the Th1 pattern and could cause undesirable pregnancy termination. The aim of the study was to evaluate the expression of HSP70 family members (Hsc70 and Hsp70), glycoprotein 96 (gp96) and their receptors CD91 and Toll like receptor 4 (TLR4) was investigated at maternal-fetal interface of the first trimester normal and pathological pregnancies (blighted ovum and missed abortion) and placental tissue at term. The mecchanisam of regulation of HSP70, gp96, CD91 and TLR4 expression was analysed by increasing concentration of Progesterone Induced Blocking Factor (PIBF), interleukin (IL)-15 and IL-2. The influence of HSP70 and gp96 on phenotype and maturation process of decidual CD1a+ dendritic cells (DC) was evaluated in vitro. Material and methods: Immunohistological and immunofluorescent labelling were performed on paraffin-embedded decidual sections of normal and pathological first trimester pregnancies (blighted ovum and missed abortion) and term placenta to detect gp96, Hsc70, Hsp70, CD91 and TLR4. The samples of normal first trimester pregnancy decidua were enzymatically digested and gradient density centrifuged to obtain the suspension of decidual mononuclear cells (DMC). DMC were stimulated with increasing concentration of PIBF, IL-15 and IL-2 and the expression of HSP70, gp96, CD91 and TLR4 was analysed by flow cytometry and RT-qPCR. The specific binding of HSP70 and gp96 was asset for receptors CD91 and TLR4 on CD1a+ DC. Flow cytometry was used to evaluate the influences of HSP70 and gp96 on the phenotype, the maturation process and the intracellular expression of cytokines and chemokines in decidual CD1a+ DC. Results: Hsc70, Hsp70, gp96, CD91 and TLR4 are present in decidual tissue of normal and pathological pregnancies first trimester (blighted ovum and missed abortion) and term placenta and they were found in glandular cells, endothelial cells, cells randomly distributed in stroma and trophoblast cells. PIBF down-regulated HSP70, gp96, CD91 and TLR4 at gene and proteins levels in DMCs. Cytokines, IL-2 and IL-15 decreased mRNA for HSP70, CD91 and TLR4, while increased mRNA for gp96. IL-2 also decreased the frequency of CD91+ and TLR4+ cells in different subpopulations of DMC. IL-15 treatment increased the frequency of CD91+ NK cells, but decreased the frequency of TLR4 expressing NK cells, T cells, immature and mature DC. HSP70 and gp96 efficiently binds CD91 and TLR4 receptors on decidual CD1a+ DC. In the presence of HSP70 and gp96 immature CD1a+ DC assume a more mature phenotype due to increased expression of CD83, HLA-DR, co-stimulatory CD80 and CD86 molecules, chemokines (CCL22 and CCL3) and cytokines (IFN-γ and IL-15). Conclusion: The presence of heat shock proteins (HSP70 and gp96) and their receptors CD91 and TLR4 at the maternal-fetal interface during normal and pathological first trimester pregnancies (blighted ovum and missed abortion) and term placenta provides a molecular basis for their interaction. The expression intensity of these molecules could be influenced by the interplay among PIBF, IL-15 and IL-2. HSP70 and gp96, as natural ligands for CD91 and TLR4 receptors induce maturation process of CD1a+ DC toward the expression of predominantly pro-inflammatory mediators, which could support a harmful immune response and adverse pregnancy outcome

COVID-19 and pregnancy: are they friends or enemies?

Abstract OBJECTIVES: Novel coronavirus disease (COVID-19) is rapidly spreading all over the world. Although in many cases the infection causes very weak symptoms, it can be severe in patient with diverse chronical diseases and immunological compromising patients. Pregnancy is a unique condition in which mother and fetus peacefully collaborate. Diverse endocrine-immune mechanisms, mostly under progesterone control work together to protect the fetus from maternal immunocompetent cell activation driven rejection. The physiological shift to Th2 dominant environment, while favourable for fetus, it makes mothers susceptible to infective pathogens, making pregnancy during COVID-19 pandemic challenging. MATERIALS AND METHODS: Studies involving COVID-19 in pregnancy and those analysing changes of immune system induced by COVID-19 were searched in databases such asPubMed, Scopus, Google Scholar and ScienceDirect. Databases were searched using a keyword COVID-19/coronavirus, that was combined with following terms: immune system, pregnancy, oestrogen, or progesterone. Search included studies published up to 01.07.2020. Almost 1,500 articles were found, but only 18 met criteria. RESULTS: Most frequent symptoms of COVID-19 in mothers infected in the late pregnancy were fever and cough accompanied with lymphopenia and elevated C-reactive protein. Mothers reported to have severe disease had comorbidities and were obese. Low rate of neonatal complications of maternal Sars-Coc-2 infection without neonatal mortality was observed

Combined upper limb and breathing exercise programme for pain management in ambulatory and non-ambulatory multiple sclerosis individuals: part II analyses from feasibility study

Abstract Purpose The present small semi-controlled feasibility study investigated a possible efficacy of a combined upper limb and breathing exercise programme in managing pain in ambulatory and non-ambulatory patients with EDSS from 0.0–8.0. Method People with MS (N = 19) were enrolled in this single-blind randomized controlled study and divided into 2 groups: exercise group (5 ambulatory, 5 non-ambulatory ; Expanded Disability Status Scale (EDSS), 1.0–8.0) and related control group that performed no exercise (4 ambulatory, 5 non-ambulatory ; EDSS, 1.0–7.5). The exercise group performed combined upper limb and breathing exercises in a group led by a physiotherapist (2 days/week, 60 min/session) accompanied by independent home exercises (3 days/week, ≥ 20 min/session). Participants underwent measures of pain level (visual analogue scale) for physical pain, functional independence of daily activities (Barthel index) and handgrip strength (HGS) for dominant (D) and non-dominant (ND) hand evaluated by a dynamometer before and after the 4-week period by the blinded assessor. Results The VAS for pain showed statistically significant group-by-time interaction only in non-ambulatory (p = .049) individuals, but with large intervention effects on both subgroups (ambulatory, p = .159 ; non-ambulatory, d = 0.97). Functional independence in daily activities (Barthel index) showed statistically non-significant group-by-time interaction in ambulatory (p = .195, d = 0.89) and non-ambulatory (p = .102, d = 1.64) individuals, but despite the absence of statistical significance, there were large intervention effects. Handgrip strength was significantly improved for both hands in ambulatory (D, p = .012 ; d = 2.07 ; ND, p = .025, d = 1.77) and only non-dominant hand in non-ambulatory individuals (D, p = .288, d = 0.83 ; ND, p = .012, d = 2.21). Conclusion This small pilot study provides preliminary proof-of-concept data supporting low-intensity upper limb and breathing exercise programme for potential reduction of pain and improvement of functional independence in both ambulatory and nonambulatory individuals with MS in a larger sample and that strengthening the upper limbs might be an additional pain relief mechanism. Trial registration NTC0322259

Raw Vegan Diet in the Context of SARS-CoV-2 Infection

Većina osoba zaraženih virusom SARS-CoV-2 razvije blagu do umjerenu koronavirusnu bolest (COVID-19), ali klinička prezentacija i opći tijek bolesti uvelike se razlikuju i uglavnom ovise o zdravstvenom stanju pacijenta prije infekcije. Određeni broj ljudi, posebno onih koji su imali teški COVID-19, ima multiorganske simptome koji traju tjednima, a u nekim slučajevima i dulje od 12 mjeseci nakon infekcije, a neki pokazuju imunofenotip koji podržava kroničnu upalu i može dovesti do razvoja autoimunosti i neuroinflamacije. Nezdrav način života i s njim povezana zdravstvena stanja, posebice pretilost, hipertenzija, dijabetes tipa 2 i hiperlipidemija, povezani su s težim oblicima bolesti COVID-19, ali podatci o utjecaju prehrane na klinički tijek bolesti još uvijek nedostaju. Sirova veganska prehrana ograničava unos nepoželjnih sastojaka hrane kao što su zasićene i nezasićene masti, natrij i dodani šećeri, a istovremeno je bogata voćem, povrćem i cjelovitim žitaricama te stoga ima mnoge pozitivne učinke na zdravlje. Svrha ovog članka je dati pregled čimbenika povezanih s težinom i ishodom bolesti COVID-19, istaknuti sve važne učinke sirove veganske prehrane na zdravlje pojedinca i raspravljati o njima u kontekstu infekcije virusom SARS-CoV-2.Most individuals infected with SARS-CoV-2 experience mild to moderate coronavirus disease 2019 (COVID-19), but the clinical presentation and general course varies widely and depends mainly on the patient's health status before the infection. Certain number of people, particularly those who had a severe COVID-19, experience multiorgan symptoms lasting weeks, months, or even years after infection and some exhibit an immunophenotype that supports chronic inflammation and may trigger autoimmunity and neuroinflammation. An unhealthy lifestyle and associated health conditions, particularly obesity, hypertension, type 2 diabetes, and hyperlipidemia, have been associated with the severity of COVID-19, but the data on the role of diet are still lacking. A raw vegan diet limits the intake of unhealthy food components such as saturated and unsaturated fats, sodium, and added sugars while being rich in fruits, vegetables, and whole grains, giving it some serious health benefits. The purpose of this review article is to give an overview of the factors associated with the severity and the outcome of COVID-19, to highlight all the important effects of raw vegan diet on individual health and to discuss them in the context of SARS-CoV-2 infection

The Role of Glycoprotein 96 in Breast Cancer

Glikoprotein 96 (gp96) član je obitelji proteina toplinskog šoka 90 koja je inače sveprisutna obitelj molekularnih šaperona uključenih u regulaciju sinteze proteina, ali i drugih esencijalnih staničnih procesa. Gp96 je smješten u lumenu endoplazmatskog retikula stanice gdje igra ključnu ulogu u homeostazi proteina, od njihove sinteze do razlaganja. Međutim, uslijed izloženosti stresornim čimbenicima koji dovode do narušavanja stanične ravnoteže, može doći do premještanja gp96 na staničnu membranu pri čemu se aktiviraju njegove dodatne funkcije, kao što su regulacija unutarstanične signalizacije, proliferacije, apoptoze te modulacija imunološkog odgovora. Pored njegove uloge u fiziološkim uvjetima, gp96 također ima i aktivnu ulogu u različitim fazama onkogeneze. U ovom preglednom članku objedinili smo dostupna saznanja o strukturi, fiziološkim te patofiziološkim ulogama gp96, prvenstveno onima u onkogenezi kod karcinoma dojke.Glycoprotein 96 (gp96) is a member of the heat shock protein 90 family, which is an ubiquitous family of molecular chaperones that are involved in the regulation of protein folding and other essential cellular activities. Residing in the lumen of the endoplasmic reticulum, gp96 plays a key role in maintaining protein homeostasis, from assemblage to degradation. However, exposure to stressful conditions that disturb cellular homeostasis may translocate gp96 to the cell surface, which implies its additional functions, such as the regulation of intracellular signalling, proliferation, and apoptosis, as well as the modulation of the immune response. Besides its roles under physiological conditions, gp96 is also included in different stages of oncogenesis. In this review, we summarised available data on the structure, physiological, and pathophysiological roles of gp96, particularly in breast cancer oncogenesis

The involvement of the progesterone receptor in PIBF and Gal‐1 expression in the mouse endometrium

Problem: The progesterone-regulated genes, PIBF and Gal‐1, are key players in the feto-maternal immunological interaction. This study aims to investigate the expression of PIBF and Gal‐1 in WT and progesterone receptor KO models as well as subsequent effects of PIBF on decidualization of stromal cells. Method of the study: PRAKO, PRBKO and PRKO BALB/c mice were used for assessing the role of PR isoforms in PIBF induction. PIBF‐ and Gal‐1 mRNA expression in the uterus was tested by real‐time PCR. The effect of PIBF on decidualization of endometrial stromal cells was verified by anti-desmin immunofluorescence. Immunohistochemistry was used for testing PIBF expression in the uterus. Gal‐1, ERα and PR positive decidual NK cells were detected by immunofluorescence. Results: PIBF mRNA was significantly increased in progesterone‐treated WT mice, but not in PRKO and PRAKO mice. PIBF protein expression was reduced in the endometria of PRKO and PRAKO, but not in PRBKO mice. During a 6‐day culture, PIBF induced decidual transformation of endometrial stromal cells. PIBF expression in the mouse uterus was highest during the implantation window, while Gal‐1 mRNA expression continuously increased between day 2.5 and day 11.5 of gestation. Decidual NK cells express Gal‐1 and ERα, but not PR at day 7.5 murine pregnancy. Conclusion: PIBF produced via engagement of PRA, is highly expressed in the endometrium during the implantation window, and plays a role in decidualization. The concerted action of PIBF and Gal‐1 might contribute to the low cytotoxic activity of decidual NK cells