Flow cytometric pitfalls in immunophenotyping of lymphomas

Flow cytometry has been introduced as the most reliable method for the analysis of lymphocyte subsets. The advantage of flow cytometry is that it enables application of virtually all monoclonal antibodies (native cells), a quantitative estimation of different lymphoid cells, and that cell can be simultaneously analysed for multiple marker expression. The usage of flow cytometers requiers considerable knowledge of physics and its technical application. Moreover, several problems arise from the complexity of the biolocal systems investigated

Korelacija med NK citolitičnim in BLT esteraznim testom pri določevanju aktivnosti NK celic stimuliranih s tumorskimi celicami

We examined the granule exocytosis in natural killer (NK) cells by measuring N-benzyloxycarboxy-L-lysine esterase activity. As a source of NK cells we usedbuffy-coat isolated NK cells or peripheral blood lymphocytes (PBL). The exocytosis was triggered by incubating cells with ionomycin/PMA or by NK cell susceptible tumour target cells K562. When we stimulated purified NK cells with tumour target cells, a close correlation (Corr=0.84) of cytolitic NK testresults and BLT test results was obtained. We may conclude that BLT test can provide a valuable tool to discriminate further NK cell deficiency in patients with low cytolitic NK test results.Z meritvami encimske aktivnosti N-benzyloxycarboxy-L-lizinske esteraze smo preiskovali eksoitozo celic NK. Celice NK smo osamili iz levkocitnega koncentrata (Buffy coat) oz. venske krvi. Eksocitozo smo sprožili s kombinacijo PMA/ionomcin ali s tumorskimi celicami K542. Ob stimulaciji celic NK s tumorskimi celicami (K542) smo opazovali značilno soodvisnost (Cor=0.H4) med rezultati citolitične aktivnosti celic NK in med izmerjenimi encimskimi aktivnosti N-benzv- loxycarboxy-L-lizinske esteraze (BLT test). Ugotavljamo, da BLT test omogoča nadrobnej5i študij dogodkov, ki se odvijajo med citotoksično aktivnostjo celic NK

Ovladavanje stresom izbjegavanjem i broj limfocita

Stress is the key psychological activator of the hypothalamic-pituitary-adrenalaxis and therefore an important risk factor for diminished immune competence. The aim of the study was to assess the connection between the strategies of coping with stress and lymphocyte counts in soldiers. Coping strategies were evaluated in 61 Slovenian Army members using the Coping Responses Inventory. White blood cell count with detailed lymphocyte analysis by use of flow cytometry was assessed in 33 soldiers. Factor analysis identified two factors of coping, i.e. avoidance coping and approach coping. Statistically significant negative correlations were recorded between avoidance strategies and monocyte, lymphocyte and T-lymphocyte concentrations. Approach strategies, which are thought to be more adaptive, did not correlate with the immune system measures. These findings support the notion that each person’s individual coping styles are reflected in their immune characteristics. We presume that avoidance coping might be an important mediating variable influencing the effects of stress on immune measures.Stres je ključni psihološki aktivator hipotalamo-pituitarno-adrenalne osi te je time važan čimbenik rizika snižavanja razine imune sposobnosti. Cilj ove studije bio je procijeniti vezu između strategija ovladavanja stresom i broja limfocita kod vojnika. U studiju je bio uključen 61 član slovenske vojske, kod kojih su se strategije ovladavanja stresom procjenjivale pomoću instrumenta poznatog kao Coping Responses Inventory. Bijela krvna slika je određena kod 33 vojnika uz podrobnu analizu limfocita pomoću protočne citometrije. Faktorska analiza je utvrdila dva čimbenika ovladavanja: ovladavanje izbjegavanjem i pristupno približavanjem. Nađene su statistički značajne negativne korelacije između strategija izbjegavanjem i koncentracija monocita, limfocita i T-limfocita. Strategije približavanjem koje se smatraju prilagodljivijima nisu korelirale s mjerama imunog sustava. Ovi nalazi govore u prilog shvaćanju prema kojem se načini ovladavanja pojedine osobe odražavaju u njenim imunim svojstvima. Pretpostavljamo da bi ovladavanje izbjegavanjem moglo biti važna posrednička varijabla koja utječe na učinke stresa na imune značajke

A Tetrahydrophthalazine Derivative »Sodium Nucleinate« Exerts a Potent Suppressive Effect upon LPS-Stimulated Mononuclear Cells in vitro and in vivo

We described the use of a new chemical substance Sodium nucleinate (SN) as an immunomodulatory substance exhibiting antiinflammatory properties. Sodium nucleinate (SN) registrated in Russian Federation as Tamerit®, is 2-amino- 1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate, derivative of well known chemical substance luminol. To comprehend the mechanisms of SN immunomodulatory activity, we examined the SN modulation of the innate inflammatory cytokine response of human PBMC stimulated with LPS in vitro. Furthermore, we studied the immunomodulatory effects of SN in mice challenged with E. coli LPS in vivo to investigate a possible novel approach to therapy of excessive inflammation that interfere with the response to endotoxin and inflammatory mediators. Our results demonstrated that SN is an efficient inhibitor of sepsis development in mice model of LPS-induced sepsis. The changes induced by SN include decreased mice plasma inflammatory cytokine production. Simmilary we demonstrated a decreased TNF-a, IFN-g and IL-6 response in human LPS-stimulated PBMNCs. SN was therefore shown to be a promising inhibitor of multiple inflammatory cytokine secretion

Tetrahydrophthalazine Derivative »Sodium Nucleinate« Exert its Anti-Inflammatory Effects through Inhibition of Oxidative Burst in Human Monocytes

We described the use of a new chemical substance Sodium nucleinate (SN) as an immunomodulatory substance exhibiting antiinflammatory properties. Sodium nucleinate (SN) registrated in Russian Federation as Tamerit®, is 2-amino- -1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate, derivative of well known chemical substance luminol. To comprehend the mechanisms of SN immunomodulatory activity, we examined the SN modulation of the oxidative burst responses of whole blood human monocytes and polimorphonuclear cells (PMC) stimulated with phorbol 12-myristate 13-acetate (PMA) or E. coli suspension in vitro. SN did not inhibit the proportion of neutrophils and monocytes phagocytosing E. coli. Oxidative burst responses of monocytes stimulated with PMA were strongly inhibited at SN concentration ranging from 10–500 mg/ml, less efficient inhibitor was SN in E. coli stimulated monocytes (inhibition range was from 50–500 mg/ml SN). SN inhibited PMC oxidative burst only in range 100–500 mg/ml SN. In conclusion, we found SN as an efficient inhibitor of oxidative burst in monocytes. Since ROS generation in monocytes/macrophages has been found to be important for LPS-driven production of several proinflammatory cytokines, SN may exsert its antiinflammatory effects through monocyte/macrophage oxidative burst inhibition

A Tetrahydrophthalazine Derivative »Sodium Nucleinate« Exerts a Potent Suppressive Effect upon LPS-Stimulated Mononuclear Cells in vitro and in vivo

We described the use of a new chemical substance Sodium nucleinate (SN) as an immunomodulatory substance exhibiting antiinflammatory properties. Sodium nucleinate (SN) registrated in Russian Federation as Tamerit®, is 2-amino- 1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate, derivative of well known chemical substance luminol. To comprehend the mechanisms of SN immunomodulatory activity, we examined the SN modulation of the innate inflammatory cytokine response of human PBMC stimulated with LPS in vitro. Furthermore, we studied the immunomodulatory effects of SN in mice challenged with E. coli LPS in vivo to investigate a possible novel approach to therapy of excessive inflammation that interfere with the response to endotoxin and inflammatory mediators. Our results demonstrated that SN is an efficient inhibitor of sepsis development in mice model of LPS-induced sepsis. The changes induced by SN include decreased mice plasma inflammatory cytokine production. Simmilary we demonstrated a decreased TNF-a, IFN-g and IL-6 response in human LPS-stimulated PBMNCs. SN was therefore shown to be a promising inhibitor of multiple inflammatory cytokine secretion

The Immune Response to Helicobacter pylori

Odgovor imunološkog sustava na infekciju bakerijom Helicobacter pylori obuhvaća različite mehanizme koji imaju pozitivan i negativan učinak na domaćina. Infekciju produljuju razni upalni i protuupalni procesi što se javljaju kao odgovor prirođene i stečene imunosti na infekciju. Iz tog je razloga teško razviti nove lijekove i učinkovita cjepiva protiv H. pylori. Usprkos mnogobrojnim postupcima imunizacije provedenim uz pomoć standardnih i rekombinantnih cjepiva (kao što su: ureaza, CagA, HP-NAP, HspA, DNA, kimerne molekule, vektori, mikrosfere), još uvijek nije pronađeno učinkovito cjepivo protiv H. pylori. Razvoj bi cjepiva trebao ići u novom smjeru, pri čemu treba dobro istražiti imunopatološke reakcije u organizmu inficiranom bakterijom H. pylori, te uvjete pri kojima dolazi do smirivanja infekcije. Stoga je važno istražiti međusoban utjecaj različitih čimbenika: upalnu reakciju sluznice želuca kao odgovor na infekciju, čimbenike virulencije bakterije H. pylori što potiču imunološki odgovor, reguliranje stečene imunosti, ali i osmisliti plan za proizvodnju novih cjepiva koja bi dovela do potpunog imunološkog odgovora tj. do potpune zaštite organizma od infekcije.The immune response to Helicobacter pylori involves different mechanisms that are both protective and damaging to the host. The innate and the adaptive immune responses lead to inflammatory as well as anti-inflammatory responses, allowing for persistence of many infections. Thus, developing new therapeutics and effective vaccines against H. pylori has proven to be arduous. Despite many immunisation experiments, using various routes of immunisation with classical as well as recombinant H. pylori vaccines (urease, CagA, HP-NAP, HspA, DNA, chimeric molecules, live vectors, microspheres), no effective vaccine is currently available for humans. New directions for successful vaccine construction should follow a profound knowledge of immunopathological events during natural H. pylori infection and factors leading to resolution of infection: mandatory is a new knowledge about the interplay of the innate response to H. pylori, mucosal inflammation, H. pylori virulence factors inducing immune responses, regulation of the adaptive responses to H. pylori as well as construction of novel vaccine platforms for achieving a broad immune response, leading to a sterilizing immunity

Inflammatory Molecules in Aqueous Humour and on Ocular Surface and Glaucoma Surgery Outcome

Purpose. To investigate the influence of inflammatory molecules in the aqueous humour and on the ocular surface on the outcome of glaucoma surgery. Methods. Thirty patients who needed antiglaucomatous surgery were included. The interleukin- (IL-) 8, IL-1β, IL-6, IL-10, tumour necrosis factor- (TNF-) α; and IL-12 were determined from aqueous humour preoperatively and the imprints of conjunctiva were analysed for expression of human leukocyte antigen- (HLA-)-DR after surgery by flow cytometry. The success of trabeculectomy was defined as intraocular pressure less than 21 mmHg without antiglaucoma medication. Results. Eyes with trabeculectomy failure at 3 months showed significantly higher TNF-α and IL-6 levels in the aqueous than eyes with successful surgery. Increased expression of HLA-DR on epithelial cells and antigen-presenting cells was not associated with the trabeculectomy outcome. Conclusions. Higher preoperative levels of TNF-α and IL-6 in aqueous humour may contribute to the development of inflammatory milieu and were associated with worse outcome of glaucoma surgery

Neutrophil and Monocyte CD64 and CD163 Expression in Critically Ill Neonates and Children with Sepsis: Comparison of Fluorescence Intensities and Calculated Indexes

Objective. To evaluate the expression of CD64 and CD163 on neutrophils and monocytes in SIRS with/without sepsis and to compare the diagnostic accuracy of CD64 and CD163 molecules expression determined as (1) mean fluorescence intensities (MFI) of CD64 and CD163; and (2) the ratio (index) of linearized MFI to the fluorescence signal of standardized beads. Patients and methods. Fifty-six critically ill neonates and children with systemic inflammatory response syndrome (SIRS) and suspected sepsis, classified into two groups: SIRS with sepsis (n = 29) and SIRS without sepsis (n = 27). Results. CD64 and CD163 MFI measured on neutrophils and monocytes were elevated in patients with SIRS with sepsis. Diagnostic accuracy of indexes was equal to diagnostic accuracy of MFI for CD64 on neutrophils (0.833 versus 0.854 for day 0 and 0.975 versus 0.983 for day 1) and monocytes (0.811 versus 0.865 for day 0 and 0.825 versus 0.858 for day 1), and CD163 on neutrophils (0.595 versus 0.655 for day 0 and 0.677 versus 0.750 for day 1), but not for CD163 on monocytes. Conclusion. CD64 MFI, CD163 MFI, CD64 indexes for neutrophils and monocytes, and CD163 index for neutrophils can all be used for discrimination of SIRS and sepsis in critically ill neonates and children. CD64 index for neutrophils, however, is superior to all other markers