Systemic and Local Increase of Granulysin Expression in Cytotoxic Lymphocytes in Severe Psoriasis.

Psoriasis is considered to be a cytokine-driven immune-mediated disease, although the cell cytotoxicity mechanisms involved remain unrecognized. Herein, we analyzed granulysin expression in different lymphocyte subsets of peripheral blood of 40 psoriatic patients (20 with severe and 20 with mild psoriasis) and seven sample of psoriatic skin. The simultaneous detection of intracellular granulysin and cell surface antigens was performed using flow cytometry in peripheral blood and immunohistochemistry in skin lesions. The frequency of granulysin+ cells, mean fluorescence intensity for granulysin, and the frequency of CD8+ T lymphocytes, NK cells, and NKT cells expressing granulysin molecules in peripheral blood were significantly higher in patients with severe psoriasis compared to mild disease and healthy individuals. These were also correlated with disease severity. Furthermore, granulysin+ cells, CD8+granulysin+ T lymphocytes, and CD56+granulysin+ NK cells were present in a higher frequency in the epidermal basal cell layer and in the dermal infiltrate of lesional skin as compared to non-lesional and healthy skin. In conclusion, granulysin+ cytotoxic cells are upregulated in blood and lesions of patients with psoriasis suggesting the involvement of granulysin mediated cytotoxicity in psoriasis pathogenesis.

Decrease of Perforin Expressing Lymphocytes after On-Pump Coronary Artery Bypass Grafting Surgery Irrespective of Carbohydrate Preoperative Oral Feeding.

BACKGROUND: Coronary artery bypass grafting (CABG) surgery continues to be the gold standard for treating the patients with coronary artery disease. CABG surgery can be performed on or off cardiopulmonary bypass, termed as on-pump or off-pump CABG, respectively. It has been shown that CABG surgery, preferably on-pump CABG surgery, leads to the changes of cell immunity during perioperative and early postoperative period. The mechanisms of regulation of the immune response in patients during and early after surgical revascularization are not fully understood. The aim of this study was to investigate the influence of carbohydrate preoperative oral feeding on frequency and perforin expression in peripheral blood lymphocytes in patients after on- or off-pump CABG surgery in early postoperative period. PATIENTS AND METHODS: In this prospective clinical study, 80 patients scheduled for CABG surgery were included in the study. The patients were randomly allocated into four groups (20 in each group): patients in Group 1 underwent on-pump CABG and did not receive carbohydrate preoperative oral feeding; patients in Group 2 underwent on-pump CABG and were preoperatively fed; patients in Group 3 underwent off-pump CABG and did not receive carbohydrate preoperative oral feeding; while patients in Group 4 underwent off-pump CABG and received carbohydrate preoperative oral feeding. Blood samples were collected immediately before (T1), 24 (T2) and 72 (T3) hours after the surgery. Peripheral blood mononuclear cells were isolated by gradient centrifugation and simultaneously labelled by antigens using fluorochrome-conjugated monoclonal antibodies. Frequency of T lymphocytes, NK and NKT cells, their subsets as well as their perforin expression were detected, and analyzed by flow cytometry. RESULTS: There was significant decrease in frequency of CD3+ and CD3+CD4+ cells, as well as perforin expressing CD3+CD8+ cells in patients who underwent on-pump CABG in comparison to patients who underwent off-pump CABG 24 hours after the surgery. Carbohydrate preoperative oral feeding did not effect changes in lymphocytes subpopulations and perforin expression at any time point. CONCLUSION: Decreases of CD3+ cells on account of CD3+CD4+ subsets, and perforin expressing cells on account of CD3+CD8+ perforin+ cells were found in patients who had undergone on-pump CABG, but not in patients who had undergone off-pump CABG surgery, irrespectively of carbohydrate preoperative oral feeding.

Postoperative Regional Analgesia Is Effective in Preserving Perforin-Expressing Lymphocytes in Patients After Total Knee Replacement.

BACKGROUND Pain and surgical stress cause a pro-inflammatory response followed by downregulation of the immune response, which can increase the incidence of postoperative complications, such as infections or prolonged wound healing. T lymphocytes and natural killer (NK) cells have cytotoxic potential and are crucial components of cellular immunity, which is important for maintenance of immune balance. The aim of this study was to analyze the effects of 3 types of postoperative analgesia on the preservation and cytotoxic potential of T lymphocytes, NK cells, and their subpopulations, as well as NKT cells, in patients after total knee replacement (TKR) to find the most effective analgesic technique for mitigating immune suppression. MATERIAL AND METHODS Forty-eight patients scheduled for TKR were randomly allocated to Group 1 (patients received epidural analgesia), Group 2 (patients received sciatic and femoral nerve block), or Group 3 (patients received multimodal systemic analgesia). Pain intensity was assessed at rest and on movement before, immediately after, and at 24 and 72 h after surgery. Blood samples were collected at the same time points and peripheral blood mononuclear cells were isolated. The frequencies of T lymphocytes, NK cells, and NKT cells, as well as their perforin expression, were simultaneously detected and analyzed by flow cytometry. RESULTS Patients in Group 1 and Group 2 experienced less severe pain than those in Group 3. The frequencies and perforin levels of T lymphocytes, their subsets, and NKT cells were significantly lower in Group 3 than in Group 1 and Group 2. CONCLUSIONS The present study confirmed that regional analgesia is more effective in maintaining cell-mediated immunity and perforin expression in peripheral blood lymphocytes in patients after TKR.

Possible role of circulating endothelial cells in patients after acute myocardial infarction.

Acute myocardial infarction (AMI) occurs as a result of insufficient myocardial perfusion leading to cell necrosis. This is most commonly due to the obstruction of the coronary artery by ruptured atherosclerotic plaque and thrombosis. Damaged ischemic and necrotic myocardial cells release pro-inflammatory substances in tissue and plasma, leading to a systemic inflammatory response. Profound systemic inflammatory response during ischemia/reperfusion injury causes disruption of endothelial glycocalyx and detachment of endothelial cells that express von Willebrant factor (vWF). We hypothesize that circulating vWF+ endothelial cells could act as antigen presenting cells which interact with T and NK cells directly, by cell to cell contact and indirectly by cytokine and chemokine secretion, leading to the immune response towards inflammation. Analyzing the frequency, phenotype and pro-inflammatory substances produced in circulating vWF positive (+) cells in patients with AMI could be beneficial to determine the severity of the pro-inflammatory response, according to the level of endothelial dysfunction in the early period of AMI. To evaluate these hypotheses, we suggest to determine frequency, phenotype, and ability of cytokine/chemokine production in circulating vWF+ endothelial cells by simultaneous surface and intracellular cell staining, and flow cytometry analysis. Secretion of pro-inflammatory cytokines and chemokines, pro-atherogenic substances and the components of glycocalyx might be measured in supernatants of magnetically separated or sorted vWF+ endothelial cells, as well as in the serum of a patient with acute AMI by enzyme linked-immunoassay tests. The interaction of increasing concentrations of isolated circulating vWF+ endothelial cells and cognate T and NK cells might be investigated by lymphocyte proliferation rate, cytotoxic mediators' expression, and cytokine production. If our hypothesis is correct, characterization of circulating vWF+ endothelial cells could grant us greater insight into their role in pathophysiology of AMI and the degree of myocardial damage.

A restrictive dose of crystalloids in patients during laparoscopic cholecystectomy is safe and cost-effective: prospective, two-arm parallel, randomized controlled trial.

PURPOSE: There are no evidence-based guidelines for volume replacement during surgical procedures such as laparoscopic cholecystectomy. However, the administration of a restrictive volume of crystalloids could be more cost-effective and safe. This trial aimed to determine the effectiveness and safety of a restrictive regimen of crystalloids in patients during laparoscopic cholecystectomy by analyzing its cost-effectiveness and 1-year morbidity rate. PATIENTS AND METHODS: In this randomized, prospective study, patients were assigned to one of three groups based on the volume of fluid administered: the restrictive group received 1 mL/kg/hr, the low liberal group received 5 mL/kg/hr, and the high liberal group received 15 mL/kg/hr of Ringer's solution intraoperatively. There were 40 patients in each group. Each patient's hemodynamic parameters and laboratory values (arterial blood gas and lactate levels) were measured together with their consumption of crystalloids, volatile anesthetics, and analgesics. RESULTS: Analysis of the hemodynamic and laboratory parameters revealed no signs of global hypoperfusion in any of the groups analyzed. There was no significant difference in the duration of surgery and anesthesia, but the consumption of crystalloids, volatile anesthetics, and opioids was significantly lower in the restrictive group, compared with the low and high liberal groups. Although there was no significant difference in the 1-year morbidity among the groups, heart failure was observed in one patient in the high liberal group in the early postoperative period. CONCLUSION: Restrictive fluid therapy during laparoscopic cholecystectomy is justified, safe, and more cost-effective than other options.

The effect of various doses of infusion solutions on the endothelial glycocalyx layer in laparoscopic cholecystectomy patients.

BACKGROUND: The endothelial glycocalyx is located on the luminal side of blood vessels and maintains vessel integrity. This study analysed how various dosages of infusion affected the secretion of atrial natriuretic peptide (ANP) and potential glycocalyx damage in patients undergoing laparoscopic cholecystectomy. We hypothesised that the liberal administration of Ringer's solution during the operation can cause iatrogenic hypervolemia with releasing of ANP and glycocalyx damage. METHODS: The study included 90 patients with American Society of Anesthesiologists' (ASA) class I and II, in good cardiopulmonary health, who were assigned to one of three groups: Restrictive group, which received 1 mL/kg/hr intraoperatively and six hours postoperatively; Low liberal group, which received 5 mL/kg/hr of Ringer's solution intraoperatively and six hours postoperatively and High liberal group, which received 15 mL/kg/hr intraoperatively and 10 mL/kg/hr six hours postoperatively. We measured patients' concentrations of glycocalyx constituents, ANP, markers of kidney and liver function, C-reactive protein (CRP), and albumine at three time points. We also measured noinvasive hemodynamics, the correlation between ANP secretion and the concentration of glycocalyx components. RESULTS: We found a significantly higher concentrations of hyaluronic acid and syndecan-1 and more ANP secretion in the High liberal group than in the other groups. We also observed a positive correlation between ANP secretion and glycocalyx constituent concentration. Markers of kidney and liver function were normal, suggesting preservation of splanchnic perfusion and global hemodynamics. CONCLUSIONS: Measuring the endothelial glycocalyx constituents in circulating blood could be a marker of intraoperative volume overload during laparoscopic operations.

Values of vanillylmandelic acid and homovanillic acid in the urine as potential prognostic biomarkers in ischaemic stroke patients.

BACKGROUND: Suitable biomarkers that have prognostic values are one of the key points of interest in ischaemic stroke. Increased sympathetic nervous system activity in ischaemic stroke causes multiple local and systemic effects that can be detrimental to the outcome. The mechanism of action is increased secretion and activity of catecholamines, whose end metabolic products are vanillylmandelic acid and homovanilic acid. Aim of our study was to determine whether these compounds can be used as potential prognostic biomarkers in ischaemic stroke, as a unique insight into the activity of the sympathetic nervous system. METHODS: Urine samples of 96 patients with ischaemic stroke and transitory ischaemic attacks were analysed. Values of vanillylmandelic and homovanillic acids in urine were tested using liquid chromatography on the first and third day post-stroke. Severity of stroke was determined using the NIHSS scale, while functional outcome was determined using the Modified Rankin Scale. RESULTS: Values of vanillylmandelic and homovanillic acids positively correlated with functional outcome of ischaemic stroke. Favorable outcomes correlated with decreased values, on contrary to increased values, which were associated with unfavourable outcomes. CONCLUSION: Determining the values of these compounds in the urine is an easily available prognostic tool for the ischaemic stroke outcome, while also influencing potential therapeutic changes.

Cytotoxic T lymphocytes as a potential brake of keratinocyte proliferation in psoriasis.

Psoriasis is a chronic papulosquamous skin disease, histologically characterized by epidermal hyperproliferation and dermal infiltration of inflammatory cells. The majority of T lymphocytes infiltrating dermis are CD4+ T lymphocytes secreting type 1 and type 17 cytokines. These cytokines are responsible for triggering keratinocyte proliferation as well as chemokine secretion and subsequent migration of other inflammatory cells in the skin. Contrarily, lymphocytes that accumulate in epidermis are mainly CD8+ T lymphocytes. According to the recent findings, these cells can also secrete type 1 and type 17 cytokines. However, it is demonstrated so far that epidermal CD8+ T lymphocytes contain higher amounts of cytolytic molecules, such as perforin, granzyme B and granulysin whose role in psoriasis pathogenesis is still unknown. Therefore, in this article we hypothesize the active involvement of cell mediated cytotoxicity in killing the proliferating keratinocytes as a mechanism of potential self-defense and possible brake in psoriatic plaque formation, maintaining skin homeostasis.

Perforin- and granulysin-mediated cytotoxicity and interleukin 15 play roles in neurocognitive impairment in patients with acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is an aggressive disease. The course of disease is regulated by pro-inflammatory agents, and malignant cell infiltration of tissues plays a deleterious role in disease progression, greatly impacting quality of life, especially in the cognitive domains. Our hypothesis is that significant serum concentrations of interleukin 15 (IL-15) are responsible for higher expression of adhesion molecules on endothelial cells of blood-brain barrier (BBB) which allow leukaemia cells and/or normal lymphocytes the infiltration into the brain. In brain tissue these cells could be stimulated to release perforin and granulysin causing induction of apoptosis in brain cells that are involved in complex neural signalling mediated by neurotransmitters, and consequent fine cognitive impairment. Such changes could be detected early, even before notable clinical psycho-neurological or radiological changes in patients with ALL. To evaluate this hypothesis we propose measuring cognitive function using Complex Reactiometer Drenovac (CRD) scores in patients with ALL. The expression of different adhesion molecules on BBB as well as presence and distribution of different lymphocytes in brain tissue will be analyzed. We will then correlate CRD scores with levels of IL-15 and the percentages of T cells, natural killer T cells, and natural killer cells expressing perforin and/or granulysin proteins. CRD is a scientifically recognised and highly sensitive psychometric laboratory test based on the complex chronometric mathematical measuring of speed of reaction to various stimuli. It provides an objective assessment of cognitive functions from the most complex mental activities to the simplest reaction reflexes. Early recognition of cognitive dysfunction might be important when selecting the most appropriate chemotherapy and/or radiotherapy regimens, and could allow for the implementation of preventive measures against further deterioration in cognitive function and quality of life in patients with ALL.

Adverse reactions and alleged allergy to local anesthetics: analysis of 331 patients.

The aim of this study was to determine the prevalence of true local anesthetic (LA) allergy among patients referred for suspected hypersensitivity and to describe the main characteristics of adverse drug reactions (ADR) induced by LA in our population. We retrospectively analyzed the medical files of patients referred to the Department of Dermatovenereology, University Hospital Center Rijeka, Rijeka, Croatia, for the investigation of LA hypersensitivity in the period between January 2000 and December 2012. A total of 331 patients underwent skin testing and, in cases of negative results, subcutaneous exposition to LA. In patients with suspected delayed reaction, patch test was performed. Altogether, 331 patients reported 419 independent ADR occurring during 346 procedures. Most commonly, patients reported having only one ADR, but 41 (12.4%) of them had two reactions, 14 (4.2%) had three, five (1.5%) had four and in one patient (0.3%) five ADR to LA were observed. The majority of reactions occurred during dental procedures when most commonly lidocaine and articaine were used. Local reactions were reported in 44 patients, whereas 490 general symptoms occurred during 375 independent ADR in 287 patients. The most common symptoms were cardiovascular system reactions in 89 patients (18.2%). Allergic reaction was detected in three patients (0.91%). One patient showed immediate-type reaction to bupivacaine and two patients had a delayed-type reaction to lidocaine. Adverse reactions to LA are common and are mostly due to their pharmacological properties and drug combinations or psychogenic origin. Allergic accidents to LA are rare.

Simultaneous appearance of cerebral venous thrombosis and subdural hematomas as rare cause of headache in puerperium following epidural analgesia: a case report.

The aim of this study is to report the first case of simultaneous appearance of cerebral venous thrombosis (CVT) and bilateral subdural hematomas (SDHs) following epidural analgesia for labor and delivery and to point out the difficulty of establishing such a diagnosis in the presence of postpartum headache. A 26-year old primigravida with a history of epilepsy received epidural analgesia for delivery. Three days after the uneventful spontaneous vaginal delivery she complained about the headache. Patient responded very well to the pain medication and oral hydration, and the headache was relieved. Ten days after the delivery, the headache reoccurred, and an epidural blood patch was performed that successfully relieved her symptom. Stronger progressive headache with nausea reappeared two days later and the parturient was readmitted to hospital. Urgent neuroimaging examinations detected CVT of right the transverse sinus, ipsilateral cortical veins, and partially occluded superior sagittal sinus, as well as bilateral subacute/chronic SDHs. The treatment of the patient with low molecular weight heparin and antiaggregation therapy was effective. In this case, the diagnosis was delayed because of atypical clinical presentation and potentially confounding events (epidural analgesia and assumption that it was a case of PDPH). It is important to carefully observe patients in such conditions and promptly conduct suitable diagnostic tests. Otherwise, unrecognized intracranial complications and delay of appropriate therapy could be life-threatening.

Expression of cytolytic protein-perforin in peripheral blood lymphocytes in severe traumatic brain injured patients.

PURPOSE: The purpose of this study was to investigate the changes of cytotoxic protein-perforin in peripheral blood lymphocytes in severe TBI patients and possible correlation between severity of TBI and perforin expression. METHODS: Flow cytometry was used for simultaneous detection of intracellular perforin and cell surface antigens of peripheral blood lymphocytes of 20 severe TBI patients on day 1, 4 and 7 after the onset of injury. Peripheral blood mononuclear cells from 20 healthy volunteers were used as control. Clinical and laboratory parameters were also recorded. RESULTS: There was a statistically significant decrease of perforin-positive lymphocytes including T, natural killer (NK) and NKT cells on day 4 as compared with day 1 after the brain injury or healthy controls. On day 7, perforin expression was restored in lymphocyte of cytotoxic phenotype (CD8(+) T lymphocytes, NK cells, and NKT cells) compared with day 1. High positive correlation was found between the severity of TBI and frequency of perforin-positive cells on day 4 when the occurrence of the intra-hospital infections was the highest. CONCLUSION: Severe TBI significantly decreases perforin expression in T lymphocytes, NK and NKT cells, which indicate a possible mechanism underlying the high susceptibility to infections.

Comparative study of frequency of different lymphocytes subpopulation in peripheral blood of patients with prostate cancer and benign prostatic hyperplasia.

PURPOSE: Benign prostatic hyperplasia (BPH) and prostate cancer (PC) are the most common urologic diseases among men over fifty and, until recently, they were considered to be caused by the impaired immune response. Despite many studies designed to investigate T-cell-based antitumor immunity, the role of innate immune cells in BPH and PC is still poorly understood. In this study the frequency of different leukocytes subpopulation in peripheral blood of BPH, PC patients and in healthy volunteers was analysed and compared. METHODS: In a cross-sectional study 60 subjects were enrolled (20 patients with BPH or with PC and 20 healthy volunteers). Peripheral blood mononuclear cells (PBMC) were isolated and the percentage of T lymphocytes, natural killer (NK) and NKT cells, as well as subsets of T lymphocytes [CD3(+)CD56(-)CD4(+), T(regs) (CD4(+)CD25(+)FoxP3(+)) and CD3(+)CD56(-)CD8(+)] and NK cells (CD3(-)CD56(+dim) and CD3(-)CD56(+bright)) were analysed by flow cytometry. Intracellular content of interleukin-4 (IL-4) and interferon gamma (IFNγ in T lymphocytes, NK and NKT cells were also detected. RESULTS: The percentage of T lymphocytes and their subsets in peripheral blood lymphocytes did not differ among investigated groups, while the frequency of Tregs was the highest in PC patients. The percentage of NK cell and their subsets did not differ among investigated groups. Negative correlation between PSA value, percentage of T lymphocytes and NK cells was observed only in PC patients. Highly positive correlation between the PSA value and the percentage of Tregs was found in PC patients. CONCLUSION: Different frequencies in distinctly lymphocyte subpopulation in peripheral blood of healthy men, BPH and PC patients could be responsible for occurrence and progression of prostatic hyperplasia or tumour. Due to the ability of tumours to suppress the cognate T cell immune response, the cells of innate immunity (NKT and Tregs) may be playing a key role in the immunopathogenesis of PC and BPH.

Short-term cytolytic mediators' expression in decidual lymphocytes is enhanced by interleukin-15.

PROBLEM: We investigated whether decidual adherent cells (DAC) and interleukin (IL)-15, in comparison to interleukin (IL)-2 affect cytolytic potential of first trimester decidual lymphocytes (DL). METHOD OF STUDY: Decidual mononuclear cells were obtained by enzymatic digestion and density gradient centrifugation. Non-adherent DL were collected after 2-hr adherence and cultured for 18 or 72 hr with: IL-15 (0.5-5 ng/mL), IL-2 (100-1000 U/mL) or both of these cytokines, DAC (ratio 3:1 and 1:1) or DAC and anti-IL-15 antibody. Perforin, Fas ligand (FasL) and granzyme B were detected at mRNA level in indicated culture conditions. Cytolytic activity of DL against K-562, P815 and P815-Fas was measured by 2-hr PKH-26 cytotoxicity assay. The dynamics of perforin protein and mRNA expression were measured in DL after a contact with K-562 targets. RESULTS: Interleukin-15 enhanced perforin, FasL and granzyme B transcription after 18-hr culture and prevented perforin protein downregulation, observed after DL culture. IL-2 had similar effects. DAC sustained perforin expression in DL and anti-IL-15 monoclonal antibody abrogated this effect. DAC increased cytotoxicity of DL against K-562 which was mediated by IL-15. CONCLUSION: Interleukin-15, probably produced by DAC, upregulates cytolytic mediators' expression and perforin-mediated cytotoxicity of DL, with equal efficiency as high concentrations of IL-2.

Perforin is required for innate and adaptive immunity induced by heat shock protein gp96.

Tumor-secreted gp96-Ig is highly immunogenic and triggers CD8 T cell-mediated tumor rejection. In vivo secreted gp96-Ig and gp96-myc cause NK activation and clonal expansion of specific CD8(+) CTL in wild-type and in Fas-ligand-deficient (gld) mice but not in perforin- (PKO) or IFN-gamma-deficient (GKO) mice. Transfer of perforin-competent NK cells restores the ability of PKO mice to clonally expand CD8 CTL in response to gp96-Ig. The data demonstrate an essential role for perforin-mediated functions in the activation of innate and adaptive immunity by heat shock protein gp96-peptide complexes. Crosspresentation of antigens by heat shock proteins seems to require a perforin-dependent positive feedback loop between NK and DC for both sustained NK activation and clonal CTL expansion. The studies also explain how depressed NK activity in patients with tumors or after viral infections could diminish CTL responses.