Investigation of Natural Killer (NK) Cell Cytotoxicity and FcγRIIIA Gene Mutationsin Patients with Low NK Cell

Doktora Tezi.Doğal öldürücü hücreler (natural killer, NK), organizmayı enfeksiyonlara ve kansere karşı savunmada önemli rol oynayan hücrelerdir. NK hücrelerini organizmada iki önemli görevi bulunmaktadır. Bunlardan birincisi, enfeksiyona neden olan mikroorganizmalara karşı direkt sitotoksik etki, ikincisi ise FcγRIIIA geni aracılığıyla gerçekleşen antikor bağımlı hücresel sitotoksisite (ADCC)'dir. NK hücrelerinin sayı veya fonksiyonunda bozukluk(lar), NK hücre eksikliği/bozukluğu (NKD) olarak adlandırılmaktadır. Bu çalışmada, klinik olarak NKD düşünülen hastalar NK hücre alt grupları, NK hücre sitotoksisitesi ve FcγRIIIA gen mutasyon(lar)u açısından araştırılması ve klinik, laboratuvar ve fonksiyonel analiz sonuçları ile FcγRIIIA gen mutasyon(lar)u arasındaki ilişkinin değerlendirilmesi amaçlandı. Çalışmaya Necmettin Erbakan Üniversitesi Meram Tıp Fakültesi Çocuk Allerji ve İmmünoloji polikliniğine ilk defa başvuran hastalar ile daha önceden takipli 10 hasta ve 7 sağlıklı bireyden oluşan kontrol grubu dahil edildi. Tüm hasta ve kontrol kan örneklerinden, periferik kan lenfosit oranları ve NK hücre alt grupları oranları akım sitometri yöntemiyle, NK hücrelerinin fonksiyonları ise sitotoksisite testi ile değerlendirildikten sonra yeni nesil dizileme yöntemi ile FcγRIIIA gen mutasyonları araştırıldı. Periferik kan akım sitometri yöntemi ile değerlendirilen NK hücre alt grup analizinde CD56brightCD16neg, CD56brightCD16int ve CD56dimCD16hi olmak üzere 3 farklı NK hücre alt grubu belirlendi. Hastaların NK hücre alt grupları kontrol grubu ile karşılaştırıldığında CD56brightCD16neg hücre oranında statistiksel olarak anlamlı bir farklılık tespit edilmedi. CD56brightCD16int ve CD56dimCD16hi hücre oranları kontrol grubu ile karşılaştırıldığında hastalarda anlamlı derecede düşük olduğu tespit edildi. NK hücre sitotoksisitesi, K562 lizisine bağlı yöntem ile değerlendirildi. NK hücre sitotoksisite analizi sonucunda hasta ile kontroller arasında K562 miktarındaki oransal olarak azalmanın istatistiksel olarak anlamlı olduğu tespit edildi (p<0,001). Hastalardaki K562 hücre azalma oranı kontrollere kıyasla istatistiksel olarak daha az azaldığı tespit edildi. Yeni nesil dizileme yöntemi ile FcγRIIIA geninin tüm exom analizinde, ekzon 4 (rs396991) ve ekzon 3 (rs10127939)'de polimorfizmler olduğu belirlendi. Ayrıca dizileme sonucunda FcγRIIIA ile FcγRIIIB ile benzerlik gösteren olası kimerik bir bölge (ekzon 1 ile ekzon 2 ile ekzon 1 arasındaki intron bölgesi) tespit edildi. Sonuç olarak bu tez çalışmasında FNKD düşünülen hastalar ileri fonksiyonel ve genetik analizler ile değerlendirildi. Hastalarda NK sayı ve fonksiyonları yönünden farklılıklar tespit edildi. FcγRIIIA geni ile ilgili tespit edilen polimorfizmler Türkiye popülasyonunda yapılmış ilk değerlendirme niteliğinde olup, NK hücre araştırmaları alanında yapılacak yeni çalışmalara ışık tutması beklenmektedir.Natural killer cells (NK) are cells that play an important role in defending the organism against infections and cancer. NK cells have two important functions in the organism. The first is the direct cytotoxic effect against microorganisms that cause infection, Secondly, it is an antibodydependent cellular cytotoxicity (ADCC) that is mediated by the FcγRIIIA gene product. Considering the important role of NK cells, disorder(s) in the NK cell number or function cause disease. This disease originating from NK cells is called NK cell deficiency / disorder (NKD). From this point, NK cell subgroups, NK cell cytotoxicity and FcγRIIIA gene mutation(s) were investigated. The aim of this study was to evaluate the relationship between clinical, laboratory and functional analysis and FcγRIIIA gene mutation(s). The study group consisted of 10 patients who were admitted to Necmettin Erbakan University Meram Medical Faculty Pediatric Allergy and Immunology Policlinic for the first time and previously monitored and consisting of 7 healthy individuals were included in the control group. All patients and controls were evaluated for the determination of peripheral blood lymphocyte ratios and NK cell subgroups and cytotoxicity tests were performed to evaluate the functions of NK cells. FcγRIIIA gene mutations were investigated by next generation sequencing method. Three different NK cell subgroups, CD56brightCD16neg, CD56brightCD16int and CD56dimCD16hi were identified in the NK cell subgroup analysis evaluated by peripheral blood flow cytometry. No statistically significant difference was found in the ratio of CD56brightCD16neg cells when NK cell subsets of the patients were compared with the control group. CD56brightCD16int and CD56dimCD16hi cell ratios were found to be significantly lower in patients compared to the control group. NK cell cytotoxicity was evaluated by K562 lysis dependent method. As a result of NK cell cytotoxicity analysis, proportional decrease of K562 amount between patient and controls was found to be statistically significant (p<0,001). It was found that the rate of K562 cell reduction in the patients decreased statistically less than the controls. Polymorphisms were detected in exon 4 (rs396991) and exon 3 (rs10127939) in the whole exom analysis of FcγRIIIA gene by new generation sequencing method. In addition, a possible chimeric region (exon 1 and exon 2 and exon 1 intron region) similar to FcγRIIIA and FcγRIIIB was determined by sequencing. In conclusion, in this study, patients with FNKD were evaluated with advanced functional and genetic analyzes. Differences were found in terms of NK number and functions. FcγRIIIA gene polymorphisms were identified related to the population of Turkey is made in the first evaluation will be done in the field of NK cell research is expected to shed light on the new study

Co-Stimulatory and Inhibitory of T Cell: CD28 Family

Hücresel bağışıklıkta rol oynayan T lenfositlerin, immün yanıt oluşturabilmesi için aktive olmaları gerekmektedir. T lenfosit aktivasyonu için iki sinyal gereklidir. Bu sinyallerden birincisi antijen tarafından sağlanır. İkinci sinyal ise, eş uyaran moleküller tarafından sağlanmaktadır. Yıllar önce T hücrelerin aktivasyonu için herhangi bir eş uyarana ihtiyaç duymadığına inanılmaktaydı. Ancak yapılan in vitro ve in vivo çalışmalar T hücrelerin aktivasyonu ve büyümesi için eş uyaranlara ihtiyaç duyduklarını göstermişlerdir. T hücre aktivasyonu ve büyümesi gerekli eş uyarılar, CD28 ailesi ve tümör nekrosis faktör (TNF) ailesi üyelerine ait olan moleküller tarafından sağlanır. CD28 ailesine ait 4 adet eş uyaran molekül tanımlanmıştır. Bunlar CD28, indüklenebilir T hücre kostimulatör sistemi (ICOS), sitotoksik T lenfosit ilişkili protein 4 (CTLA-4, CD152) ve programlanmış hücre ölümü proteini 1 (PD-1) molekülleridir. CD28 ve ICOS T hücre aktivasyonunda pozitif etkili iken, CTLA-4 ve PD-1 molekülleri negatif etki göstermektedir. Bu derlemede CD28 ailesine ait eş uyaran moleküller, bu moleküllere ait ligandlar ve eş uyaran moleküllerin ligandlarla etkileşimleri tartışılmaktadır.T cells that play role in cell-mediated immunity must be activated in order to generate immune responses. The two signals are required for T cell activation. The first of these signals is provided by the antigen. The second signal is provided by the co-stimulatory molecules. For many years it was believed that T cells do not require co-stimulation for activation. However, in vitro and in vivo studies have shown that T cells need the co-stimulatory molecules for activation and expansion. Co-stimulation for T-cell activation and growth is provided by molecules of CD28 and the tumor necrosis factor (TNF) family members. Four co-stimulatory molecules belonging to the CD28 family have been identified. These family members are CD28, inducible co-stimulator (ICOS), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) and programmed cell death protein 1 (PD-1). Although CD28 and ICOS showed positive effects, CTLA4 and PD-1 molecule have a negative effect on T cell activation. In this review, we discussed co-stimulatory molecules belonging to the CD28 family, the ligands of these molecules, and the interactions of co-stimulatory molecules with ligands

Outcome of Thrombotic Thrombocytopenic Purpura Patients: A Single-Center Experience

WOS: 000482629200016PubMed: 30905143

Clinical Evaluation of Acute Pancreatitis Caused by SARS-CoV-2 Virus Infection

Introduction. Coronavirus 2019 disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has spread to more than 200 countries worldwide. We aimed to present acute pancreatitis (AP) cases caused by SARS-CoV-2 viral infection. Methods. The study was conducted retrospectively between April 2020 and June 2020 in Necmettin Erbakan University Meram, Medical Faculty Hospital, and 150 hospitalized patients diagnosed with COVID-19 were included. The degree of acute pancreatitis was determined according to the Atlanta classification. Organ failures of the patients were evaluated in terms of respiratory, cardiovascular, and nephrology according to the modified Marshall scoring (MMS) system, and CTSI (Balthazar score) and Imrie score were determined. Modified Marshall score≥2 was considered organ failure. Results. A total of 29 patients were diagnosed with acute pancreatitis. All 29 patients with pancreatitis had respiratory failure during hospitalization. After the diagnosis of pancreatitis, there was no change in respiratory failure. According to the Atlanta classification, 19 patients had mild AP and 10 patients had moderate AP. Patients with acute pancreatitis were scored according to the CTSI (Balthazar score), and there were no patients with ≥6 severe pancreatitis. The CTSI score of 4 patients was 3. In addition, the Imrie score of the patients was determined and 8 patients with Imrie score≥3 were identified. Conclusion. The rate of pancreatic damage in SARS-CoV-2 infection was found to be 19% (n=29) in our study. In our study, we highlight acute pancreatitis as a complication associated with COVID-19 and the importance of pancreatic evaluation in patients with COVID-19 and abdominal pain is demonstrated

The effects of acute high intensity interval training on hematological parameters and neutrophils to lymphocytes ratio in elite taekwondo athletes according to gender

Introduction and aim. Intense taekwondo (TKD) training, it is important to know the exercise-induced hematological and inflammatory conditions and to develop conditions suitable for physiological needs. The aim of study is to investigate the effects of TKD-specific training containing a high-intensity interval training (HIIT) component hematological parameters and on systemic inflammatory biomarkers between gender. Material and methods. The research was carried out with twenty-four elite TKD athletes (12 female, 12 male). 90 minutes of TKD-specific unit training, including 50 minutes of HIIT component was applied to the athletes. Hematological parameters included erythrocytes, platelets, leukocytes and their subgroups and inflammatory biomarkers. Results. With the effect of TKD-specific HIIT, erythrocytes and hematocrit values decreased regardless of gender (p=0.003, p<0.001, respectively). Platelet values decreased in male and increased in female (p=0.637). White blood cells and neutrophil (p<0.001) and inflammatory biomarkers neutrophils-to-lymphocytes ratio (NLR) and platelet-to-lymphocytes ratio PLR (p[removed

Effectiveness and Safety of Autologous Stem Cell Mobilization with Granulocyte Colony Stimulating Factor in an Inpatient or Outpatient Setting

Aim:Autologous hematopoietic stem cell transplantation is the most frequently used treatment method in the treatment of lymphoma and myeloma patients. To apply this treatment method, first of all, a sufficient number of stem cells must be collected from the patient. With the development of apheresis methods and safe, effective mobilization methods, it is now possible to collect stem cells in an outpatient manner. In our study, we aimed to compare the efficacy and safety of outpatient based mobilization versus inpatient based mobilization of hematopoietic stem cells with granulocyte-colony stimulating factor (G-CSF) alone in patients with myeloma and lymphoma.Materials and Methods:A total of 89 patients, including 54 patients who underwent outpatient and 35 patients who underwent inpatient based mobilization of stem cells with G-CSF alone were included in the study. Outpatient and inpatient based mobilization groups were compared in terms of efficacy and safety. Statistical analyses were performed with Jamovi 1.2.27 software. The Mann-Whitney U and chi-square tests were used to examine the differences. MANCOVA was used for univariate and multivariate statistical analysis of factors influencing mobilization.Results:Three leukaphereses resulted in the collection of a mean 9.73x106/kg (4.5-16.5) CD34+ cells in the outpatient based mobilization group and a mean 11.8x106/kg (3.56-59) CD34+ cells in the inpatient based mobilization group (p=0.14). Life-threatening side effects were not observed in any of the patients. Grade 1, 2 side effects were observed and there was no significant statistical difference between the two groups.Conclusion:In this study, we found no significant difference in terms of efficacy and safety between the outpatient and the inpatient based mobilization group patients with myeloma and lymphoma who were mobilized with G-CSF. The results of our study show that outpatient based mobilization can be effectively and safely performed with g-csf, especially in patients who need autologous transplantation and avoid hospitalization, as in the current Coronavirus disease-2019 pandemic

Covid-19 hastalarında konvelesan plazma tedavisinin etkinliği

Convalescent plasma (CP) therapy has been used for treatment, although it has not been Corona Virus 2019 Disease (COVID-19) specific antiviral agent so far. However, the effectiveness of CP treatment on prognosis and mortality is still a matter of debate. In this study, we aimed to share our experiences about the effectiveness of CP treatment in COVID-19. Patients and Methods: The study was conducted in 126 patients diagnosed with COVID-19 who received CP treatment in addition to standard treatment between May 2020 and February 2021. 126 patients were divided into two groups as those who underwent SP within the first five days (Group A) and after five days (Group B). The patients in these two groups were evaluated in terms of laboratory parameters, clinical and mortality. Results: A total of 126 patients were identified (86 patients in Group A and 40 patients in Group B). 119 (94.4%) patients were discharged with recovery, 7 (5.5%) patients died. The mean days of hospitalization were found to be 11.4±0.7 in Group A and 18.4±1.7 in Group B (p<0.001). Treatment-related lymphocyte, PLT, fibrinogen and CRP main effect of change was significant (p<0.001). However, the results were marginally significant when the two groups were compared in terms of D-dimer. When the simple effect is evaluated; Group A as not significant, while group B was significant. Starting CP treatment 5 days before or 5 days later did not change the laboratory parameters. However, D-dimer was marginally significant (p=0.058). Conclusion: In our study, it was shown that early initiation of CP treatment reduced the hospitalization, but had no effect on mortality and laboratory parameters.Corona Virüs 2019 Hastalığı (COVID-19)’nda şu ana kadar spesifik antiviral ajan olmamasına rağmen tedavi için konvelesan plazma (CP) tedavisi tedavi için kullanılmıştır. Ancak CP tedavisinin prognoz ve mortalite üzerindeki etkinliği halen tartışma konusudur. Bu çalışmada COVID-19 hastalığında CP tedavisinin etkinliğine ilişkin deneyimlerimizin paylaşması amaçlandı. Hastalar ve Yöntem: Çalışma Mayıs 2020-Şubat 2021 tarihleri arasında standart tedaviye ek olarak CP tedavisi alan 126 COVID-19 tanılı hastada gerçekleştirildi. 126 hasta ilk beş gün içinde (Grup A) ve beş günden sonra (Grup B) CP uygulananlar olarak iki gruba ayrıldı. Bu iki gruptaki hastalar laboratuvar parametreleri, klinik bulgular ve mortalite açısından değerlendirildi. Bulgular: Toplam 126 hasta Grup A'da 86 hasta ve Grup B'de 40 hasta) tespit edildi. 119 (%94.4) hasta şifa ile taburcu olurken 7 (%5,5) hasta kaybedildi. Ortalama hastane yatış süresi Grup A'da 11.4±0.7, Grup B'de 18.4±1.7 gün olarak bulundu (p<0,001). Lenfosit, PLT, fibrinojen ve CRP’nin tedaviye bağlı ana değişim etkisi istatistiksel olarak anlamlıydı (p<0.001). Ancak, iki grup D-dimer açısından karşılaştırıldığında sonuçlar marjinal olarak anlamlıydı. Basit etki değerlendirildiğinde; Grup A’daki değişim anlamlı değilken, Grup B’deki değişim anlamlıydı. CP tedavisine 5 gün önce veya 5 gün sonra başlanması laboratuvar parametrelerini değiştirmedi. Ancak, D-dimer ’daki değişim marjinal olarak anlamlıydı (p=0.058). Sonuç: Çalışmamızda CP tedavisine erken başlamanın hastanede kalış süresini azalttığı ancak mortalite ve laboratuvar parametreleri üzerine etkisinin olmadığı gösterildi

Effective anticancer agents based-on two Pillar[5]arene derivatives for pancreas cancer cell lines: synthesis, apoptotic effect, caspase pathway

This study aimed to evaluate the possible anticancer effects of two different pillar[5]arene derivatives (5Q-[P5] and 10Q-P[5]) on two different pancreatic cancer cell lines in vitro. For this purpose, changes in the expression of major genes that play a role in apoptosis and caspase pathways were investigated. Panc-1 and BxPC-3 cell lines were used in the study and the cytotoxic dose of pillar[5]arenes was determined by the MTT method. Changes in gene expression after pillar[5]arenes treatment were evaluated by real-time polymerase chain reaction (qPCR). Apoptosis was studied by flow cytometry. As a result of analysis, it was determined that proapoptotic genes and genes involved in major caspase activation were upregulated and antiapoptotic genes were down-regulated in Panc-1 cell line treated with pillar[5]arenes. Flow cytometric apoptosis analysis also showed an increased apoptosis rate in this cell line. On the contrary, although MTT analysis showed cytotoxic effect in BxPC-3 cell line treated with two pillar[5]arene derivatives, the apoptosis pathway was not active. This suggested that it may activate different death pathways for BxPC-3 cell line. Thus, it was first determined that the pillar[5]arene derivatives reduced cancer cell proliferation on pancreatic cancer cells

Impacts of potential anticancer agents based on pillar[5]arene for head and neck squamous cell carcinoma cells

PMID: 37126106WOS:000979964000002This study was conducted to investigate impacts of potential anticancer (associated with apoptosis and caspase pathways) of two newly synthesized derivatives of pillar[5]arene, named as d-Q-P5 and p-Q-P5, on Squamous cell carcinomas of the head and neck (HNSCC) cells. The MTT method was used to determine the IC50 doses of the derivatives on HNSCC cells, and the changes in gene expression were analyzed by real-time polymerase chain reaction (qPCR). The apoptosis change was confirmed by flow cytometry analysis. The results showed that the d-Q-P5 and p-Q-P5 effectively inhibited the proliferation of the cells by upregulating proapoptotic genes (Bax, Bad, p53, Bak, and Apaf-1) and genes involved in the caspase pathway (Casp2, Casp3, and Casp9), while downregulating the antiapoptotic gene (Bcl-2). This study is the first to demonstrate the potential anticancer effects of these two agents on HNSCC cells by positively regulating apoptosis gene expression

A family screening of CD19 gene mutation by PCR-RFLP

Introduction and aim. Mutation(s) in the gene encoding the CD19 molecule affect CD19 protein expression and primary immunodeficiency (PID) occurs. The PCR-RFLP method, which is faster and cheaper than other mutation detection methods, is rarely used in the diagnosis of PID. The study aimed to genetically identify CD19 deficiency, which is a PID, using the PCR-RFLP method. Material and methods. A total of 8 patients and two healthy controls were included in the study and the relevant region genotypes in the CD19 gene were determined by performing PCR-RFLP analysis. Results. The index case, newborn baby and mother were also included in the study. It was determined that the index case (P6) was homozygous mutant, the newborn baby (P7) and mother (P8) had heterozygous genotype. Based on this situation, one child (P1) was found to be homozygous mutant, mother (P2), father (P3) and other children (P4 and P5) had heterozygous genotype in the family, which was determined to be related to the first case. Conclusion. In our study, it has been shown that PCR-RFLP is a method that can be used in the diagnosis of PID by determining genotypes using PCR-RFLP, and especially in terms of rapid genetic testing of family screenings