Total costs of inpatient treatment for COVID-19 in a tertiary hospital in Serbia.

BACKGROUND: Our study aimed to identify the total costs of inpatient treatment for coronavirus disease 2019 (COVID-19) in a tertiary institution in Serbia, an upper-middle-income country in Southeast Europe. METHODS: An observational, retrospective, cost-of-illness study was performed from the perspective of the National Health Insurance Fund and included a cohort of 78 females and 118 males admitted to the COVID-19 ward units of a tertiary center during the first wave of the pandemic. RESULTS: The median of the total costs in the non-survivors subgroup (n =43) was 3,279.16 Euros [interquartile range (IQR): 4,023.34; range: 355.20-9,909.61) which is higher than in the survivors (n =153) subgroup 747.10 Euros (IQR: 1,088.21; 46.71-3,265.91). The cut-off value of 156.46 Euros regarding the total costs per day was estimated to have 95.3 % sensitivity and 91.5 % specificity for predicting patients' dismal prognosis, with the area under the curve (AUC) of 0.968 (95 % confidence interval: 0.940-0.996, p <0.001). CONCLUSIONS: Direct medical inpatient treatment costs for COVID-19 represent a significant economic burden. The link between increased costs and an ultimate unfavorable outcome should be further explored.HIPPOKRATIA 2022, 26 (2):62-69.

Prevalence and diagnostic significance of specific IgA and anti-heat shock protein 60 Chlamydia trachomatis antibodies in subfertile women.

The objective of the present study was to evaluate the clinical usefulness of the simultaneous measurement of three serological markers of chlamydial infection in women with tubal factor infertility (TFI) and spontaneous miscarriage. Serum was collected from 87 patients (33 with TFI and 54 with spontaneous miscarriage) and analyzed for the presence of IgG and IgA antibodies against Chlamydia trachomatis MOMP antigen (Dia.Pro) and IgG antibodies to chlamydial heat shock protein 60 (cHSP60) antigen (Medac). We determined a high degree (64.5 %) of seropositivity against chlamydial antigens in our study population. The prevalence of persistent chlamydial infection has tended to be higher in the group of patients with TFI (41.4 %) than in patients with spontaneous miscarriage (21.3 %). The serum level of IgA, as a marker of active infection, was statistically higher in the TFI group with persistent infection than in the corresponding spontaneous miscarriage group (p = 0.008), while the serum level of IgG showed no statistically significant differences compared with the spontaneous miscarriage group with persistent infection (p = 0.227). Also, using the receiver operating characteristic (ROC) curve, we found that the serum level of IgA has the ability to discriminate patients with persistent chlamydial infection between the TFI and miscarriage groups, with a sensitivity and specificity of 74.3 % and 71.4 %, respectively. To the best of our knowledge, the present study is the first study which, besides the already confirmed linkage between serologic evidence of persistent chlamydial infection and TFI, also confirmed associations between spontaneous miscarriage and serologic evidence of persistent chlamydial infection.

Antioxidant enzymes activities and plasma levels of oxidative stress markers in B-chronic lymphocytic leukemia patients.

PURPOSE: Overproduction of reactive oxygen species (ROS) intermediates above the functional capability of cellular antioxidants may result in instability of important macromolecules and represents the molecular basis of many diseases including inflammation processes, cardiovascular alterations, cancer etc. The purpose of this study was to determine plasma level of superoxide anion, hydrogen-peroxide and malondialdehyde (MDA) as markers of oxidative stress and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) as antioxidant enzymes in B-chronic lymphocytic leukemia (B-CLL) patients. METHODS: The study included 29 untreated B-CLL patients in stage A, and 21 in stages B and C, classified according to the Binet system; 31 healthy volunteers formed the control group. After centrifugation of heparinized peripheral blood, plasma levels of all investigated parameters were determined using spectrophotometric methods. RESULTS: Plasma CAT activity was increased in B-CLL patients compared with control subjects; also, progression of disease was related with significantly higher plasma activity of CAT. Also, B-CLL patients showed significantly higher plasma concentration of MDA compared with controls. No statistically significant differences of superoxide anion and hydrogen peroxide as well as plasma activity of SOD and GPx between the tested groups were noted. CONCLUSION: Increase of CAT activity in B-CLL patients indicates that there is stimulation of the antioxidant enzyme system, while the increase of MDA concentration shows increased lipid peroxidation level. According to these results it could be concluded that an imbalance exists between oxidants and antioxidants in the plasma of B-CLL patients.

In vitro induction of apoptotic cell death in chronic lymphocytic leukemia by two natural products: preliminary study.

PURPOSE: B cell chronic lymphocytic leukemia (B-CLL) is an neoplastic disorder characterized by alterations in the pathways of programmed cell death (apoptosis). Deregulation of apoptosis pathways also contributes to chemoresistance of B-CLL cells. Therefore, it is not surprising that induction and acceleration of apoptosis represent key point in novel B-CLL therapeutic protocols. The present study was designed to investigate the effects of two natural products, Immunarc forte and Korbazol on the in vitro survival of leukemic cells. METHODS: peripheral blood mononuclear cells (PBMC) from 20 B-CLL patients and 20 healthy donors were used for cytotoxicity studies. Cytotoxic activity of the tested products were assessed by the MTT colorimetric assay and the type of cell death was determined by flow cytometry. RESULTS: we found that Korbazol was selectively cytotoxic against B-CLL cells, but the cytotoxic activity of Immunarc forte was much weaker. Of note, synergy was shown between these two drugs, and this effect was also selective, without affecting the normal mononuclear cells. According to Annexin-V binding, Korbazol and Immunarc forte induced apoptotic type of cell death in B-CLL cells. Moreover, treatment with Korbazol, but not with Immunarc forte, decreased spontaneous apoptosis in cultured normal polymorphonuclear cells. CONCLUSION: our findings imply that Korbazol is as potential therapeutic agent that induces apoptosis of B-CLL cells. The resistance of normal mononuclear cells and anti-apoptotic effects on normal polymorphonuclear cells, as well as its ability to synergize with Immunarc forte, warrants further investigation and supports their therapeutic application in the treatment of B-CLL.

Endoplasmic reticulum stress associated with caspases-4 and -2 mediates korbazol-induced B-chronic lymphocytic leukemia cell apoptosis.

PURPOSE: B-cell chronic lymphocytic leukemia (B-CLL) is an incurable disease that rapidly develops drug resistance. Therefore there is a need for identifying new agents that will improve the therapeutic outcome. Korbazol is a natural product known to exert cytotoxic effect on the in vitro survival of leukemic cells. The aim of this study was to investigate the mechanism of korbazol-induced apoptosis in B-CLL leukemic cells. METHODS: peripheral blood mononuclear cells from 10 B-CLL patients were used for assessing the effect of caspase inhibitors and chelator of intracellular Ca(2)+. RESULTS: cell death rate induced by the tested compound was decreased with the caspase-3 inhibitor Ac-DEVD-CHO, and the inhibitors of caspase-2 (Z-VDVAD-FMK) and -4 (ZYVAD- FMK), but not with the caspase-9 inhibitor z-LEHD-FMK and caspase-8 inhibitor z-IETD-FMK. No significant release of cytochrome C (cyt C) from mitochondria to the cytosol of B-CLL cells treated with korbazol was observed. Moreover, chelating of intracellular Ca(2)+ with BAPTA-AM almost completely abolished the cytotoxic effect of korbazol. CONCLUSION: engagement of caspases-2 and -4 and mobilization of intracellular Ca(2)+ indicate involvement of endoplasmic reticulum (ER) stress in apoptosis induced by korbazol.

Oxidative stress accelerates spontaneous apoptosis of B-chronic lymphocytic leukemia lymphocytes.

PURPOSE: B-chronic lymphocytic leukemia (B-CLL) is characterized by the progressive accumulation of small immature B lymphocytes which do not undergo apoptosis due to an underlying defect. One potential mechanism of defective apoptosis could be irregular oxidative stress. The goal of our investigation was to determine in vitro production of oxidative stress markers by lymphocytes of B-CLL patients. PATIENTS AND METHODS: 30 untreated stage A B-CLL patients, as well as 20 stage B and C patients and 30 healthy volunteers as a control group were examined. Nitric oxide (NO), superoxide anion, hydrogen peroxide and malondialdehyde (MDA) were measured by spectrophotometry in supernatants of lymphocytes cultures of all 3 investigational groups. The method applied for detecting apoptosis was fluorescence microscopic analysis using acridine orange/ethidium bromide (AO/EB) double staining. RESULTS: In vitro lymphocyte production of superoxide anion, hydrogen peroxide and MDA was increased in B-CLL patients, while there were no statistical significantly differences of NO production among the tested groups. Compared with the spontaneous apoptosis observed in control subjects lymphocytes, B-CLL lymphocytes showed increased percentages of apoptotic cells after incubation for 24 h. Disease progression was not followed with significant differences in spontaneous apoptosis of B-CLL lymphocytes. CONCLUSION: This intensive oxidative stress markers production in cultures of B-CLL lymphocytes could be one of the potential mechanisms in the pathogenesis of abnormal apoptosis.

Neuron specific enolase tissue expression as a prognostic factor in advanced non small cell lung cancer.

PURPOSE: To determine the frequency of neuron specific enolase (NSE) tissue expression and its possible influence on survival of patients treated for advanced non small cell lung cancer (NSCLC). PATIENTS AND METHODS: We studied 158 patients with histological diagnosis of NSCLC (stage III/IV). Combined chemoradiotherapy was used in stage III disease (without pleural effusion), whereas chemotherapy only was used in stage III (with pleural effusion) as well as in stage IV disease. Immunohistochemical analysis of NSE expression was determined using antibodies to NSE (DAKO, Den). One- and 2-year overall survival were assessed. RESULTS: 45 (28.5%) patients had positive NSE expression. The most frequent NSE expression was seen in 6/9 (66.7%) patients with large cell carcinoma and in 23/50 (46%) with adenocarcinoma. One- and 2-year survival rates were 70% and 30% in the group of patients with positive NSE expression compared to 26% and 3% in the group with negative NSE expression (p=0.000). The median survival time was 16.4 vs. 11.4 months in the groups of patients with and without NSE expression, respectively (p < 0.001). One-and 2-year survival rate was higher in NSCLC patients with more than 50% of NSE positive cancer cells (p=0.0004 and 0.000, respectively). CONCLUSION: A total of 28.5% of advanced NSCLC patients had positive NSE expression. Median 1- and 2-year survival time was significantly longer in patients with positive NSE expression.

HER-2 expression in uterine cervix carcinogenesis.

PURPOSE: To assess the expression and clinical significance of HER-2 protooncogene in the uterine cervix carcinogenesis. PATIENTS AND METHODS: We examined 69 tissue samples of low grade cervical squamous intraepithelial lesions (SIL) (n=16), high grade SIL (n=11) portio vaginalis uteri (PVU) carcinoma in situ (n=11) and PVU invasive carcinoma, stage IA-IIA (n=13; study group) and 18 samples without SIL or malignancy (control group). The expression of HER-2 was detected immunohistochemically using a monoclonal antibody. Fisher's exact test was used to assess statistical significance. By establishing sensitivity and specificity of the test, the level of reliability of these analyses was determined as a possible screening method for early detection of changes in the uterine cervix. RESULTS: Overexpression of HER-2 was found to increase in direct relation to the grade of the cervical lesions. Statistically significant difference was found in the frequency of overexpression in patients with high grade SIL, PVU carcinoma in situ and PVU invasive carcinoma compared with the control group. High sensitivity was of great diagnostic significance for the detection of these types of changes in the uterine cervix. On the basis of high predictive values it can be concluded that in patients with HER-2 overexpression there is a great possibility that they have premalignant or malignant changes in the uterine cervix. CONCLUSION: Our results indicate that overexpression of HER-2 oncogene may play an important role in cervical carcinogenesis. However, more extensive series of samples is required to establish the prognostic significance of HER- 2 in cervical carcinogenesis.

Relationship of serum levels of tumor markers with tissue expression of gene products in ovarian carcinoma.

PURPOSE: The aim of this prospective study was to determine serum levels and tissue expression of CA125, CA 15-3, p53, HER-2 and nm23 tumor markers, which are used in the detection and follow up of patients with ovarian carcinoma. PATIENTS AND METHODS: 19 patients with malignant and benign ovarian tumors were included in this study. Serum levels of CA125, CA 15-3 and p53 tumor markers were detected in preoperative and postoperative blood samples using ELISA technique. Tissue expression of p53, HER-2 and nm23 were examined using immunohistochemistry. RESULTS: All serum tumor markers were elevated in patients with ovarian carcinoma. Serum level of CA 15-3 was increased in patients with ovarian carcinoma (median 48.33 U/ml, normal range 0-36), while it was normal in patients with benign ovarian tumors (median 20.67 U/ml; p >0.05). CA125 serum values were strikingly increased in ovarian carcinoma (median 264.16 IU/ml, normal range 0-35) and benign ovarian tumors (median 119.59 IU/ml; p <0.05). Serum levels of p53 in patients with ovarian carcinoma were increased (median 0.69 U/ml, normal range 0-0.50) compared to patients with benign tumors (0.32 U/ml; p <0.05). Histological HER-2 overexpression was detected in 7 cases, including 4 with strong (score 3+ and 2+) and 3 with weak or no HER-2 expression (score 1+ and 0) in ovarian carcinoma tissue; in benign tumors HER-2 overexpression was detected in 1 case (p >0.05). Strong overexpression of p53 was detected in 3 cases with malignant and none with benign tumors (p >0.05); and strong overexpression of nm23 was detected in 5 cases with malignant and 2 with benign tumors (p >0.05). CONCLUSION: Serum levels of CA125, CA 15-3 and p53 are strikingly increased, as well as the expression of HER-2 and p53 in carcinomatous tissue. Detection and analysis of multiple tumor-specific markers in serum and tissue can give useful clinical information for the management of ovarian carcinoma and can also improve the sensitivity and specificity of these markers.

In vitro assay for the quantitative measurement of apoptotic lymphocytes phagocytosis by peripheral blood monocytes.

Currently used assays for the quantification of apoptotic cells uptake by phagocytes have several methodological problems. Our assay overcomes some of these problems. As a source of apoptotic cells we used peripheral blood lymphocytes obtained from the patients with chronic lymphoblast leukaemia. Apoptosis was induced by incubating cells with cycloheximide for up to 24 h. The assay was performed in suspension of peripheral blood mononuclear cells. For the visualisation of the phagocytes and phagocyted cells and discrimination of phagocyted from bound apoptotic cells we used Acridine orange/Ethidium bromide double staining. Here we offer a simple test which enables reliable measurement and it can show the difference of phagocytic potential between different individuals.

Preliminary study of mononuclear phagocytosis during breast cancer therapy.

PURPOSE: To evaluate the eventual changes in the number and phagocytic functions of blood monocytes in breast cancer patients during surgical treatment and chemotherapy. MATERIALS AND METHODS: The absolute and relative number of peripheral blood leukocytes and monocyte phagocytic functions were determined at the time of diagnosis (I), following surgery (II), during (III) and after chemotherapy (IV) in 30 patients diagnosed with breast cancer. The control group consisted of 30 age-matched healthy women. RESULTS: The mean number of monocytes was significantly lower in cancer patients at diagnosis, while they increased following surgery reaching the control values. There were no postchemotherapy changes in the number of monocytes. Monocyte phagocytic activity was decreased at the time of diagnosis. Following surgery, the capacity of phagocytosis (CP) recovered to normal values, but the index of phagocytosis (IP) remained decreased. During and after chemotherapy, as well as one year after surgery, the IP still remained decreased. CONCLUSION: Our results showed that some properties of monocytes' phagocytic activity in cancer patients were decreased at diagnosis, returning back to normal range after surgical therapy. However, time is needed to confirm whether the alteration of IP may provide additional information when monitoring breast cancer patients.

Serum HER2 and CA 15-3 in breast cancer patients.

PURPOSE: The HER2 oncogene encodes a transmembrane tyrosine kinase receptor. This molecule could be a new marker for prognosis and response to therapy in patients with advanced breast cancer. However, the extracellular domain of c-erbB-2 (HER2) transmembrane receptor undergoes proteolytic cleavage from the fulllength protein by metalloproteases, and is shed into the blood as a circulating antigen. To determine the clinical utility of this oncoprotein, the soluble form of HER2 was assayed in the serum of breast cancer patients. PATIENTS AND METHODS: Serum levels of breast carcinoma antigens CA 15-3 and HER2 were determined in 60 patients, 40 with localized (group A) and 20 with metastatic (group B) breast carcinoma. CA 15-3 measurements served as "gold standard" to which HER2 diagnostic and/or prognostic value was compared. Sera from 10 healthy women served as controls. RESULTS: Serum levels of the tested tumor markers HER2 and CA 15-3 were significantly higher in cancer patients compared to controls. CA 15-3 correlated with bulky initial tumor, whereas HER2 showed no differences between healthy individuals and group A patients. The serum levels of the tested markers in group B patients were significantly higher (p <0.001) than the serum levels of patients in group A. Striking increase in serum levels of HER2 was found in 52.7% and CA 15-3 in 52.9% of patients with metastatic cancer. A combination of markers was more sensitive than using one marker alone. In this regard, 90% of the patients with metastasis had at least one of the markers increased. CONCLUSION: The results of this study suggest that the HER2 oncoprotein may be potentially useful in detecting recurrence of breast cancer. However, to improve sensitivity and specificity in the diagnosis and monitoring of breast cancer, the use of multiple tumor markers should be employed.

Blood monocytes and tumor-associated macrophages in human cancer: differences in activation levels.

This study was performed to investigate functional properties of mononuclear phagocytes isolated from ascitic fluid in patients with peritoneal carcinomatosis (PC), and potential immunomodulatory effects of soluble factors produced or induced by human metastatic malignant cells. Phagocytic activity and nitric oxide production of peripheral blood monocytes (PBMo) and tumor-associated macrophages (TAM) or peritoneal macrophages (PEM) were synchronously examined in cancer patients and control individuals. Our results showed that contrary to peripheral blood monocytes, where phagocytic activity was not altered, TAM had impaired phagocytic activity. Moreover, dilutions of crude supernatant from short-term cultures of the peritoneal cells obtained from ascitic fluid of patient with PC, cause a significant, dose dependent inhibition of control PBMo and PEM phagocytosis, comparable to those in TAM, indicating that a soluble factor(s) plays a prominent role in this alteration. Next, we investigated the potential of cancer patients mononuclear phagocytes to produce nitric oxide (NO). It was found that TAM produce fourfold lower levels of NO than PEM from control subject, whereas monocytes produce NO at levels comparable to those of corresponding controls. These data support the hypothesis that depressed TAM function may contribute to the mechanisms of tumor escape from immune destruction.

Systemic response of peripheral blood leukocytes and their phagocytic activity during acute myocardial infarction.

OBJECTIVES: To determine changes in leukocyte counts and phagocytic activity of peripheral blood mononuclear (MN) and polymorphonuclear (PMN) cells as potential cellular markers of systemic immunological events in acute myocardial infarction (AMI). PATIENTS AND METHODS: Thirty patients with a first AMI and 30 healthy volunteers were examined. Immunological analyses were performed at admission and repeated at one and seven days after the acute event. MN and PMN cells were obtained from heparinized whole blood after centrifugation and separation on a density gradient, and incubated with a fixed number of heat-inactivated and labelled yeast particles. Total leukocyte counts, leukocyte populations and some parameters of phagocytic activity were determined: percentage phagocytosis, phagocytic index, absolute phagocytic index, count of phagocytes in a fixed volume of peripheral blood (CP) and phagocytic capacity. RESULTS: Patients with AMI had increased total leukocyte counts accompanied by increased PMN counts, while there were no significant differences in total MN count and MN populations. Except for the phagocytic index, all phagocytic parameters of MN and PMN cells were increased in patients with AMI at admission and on the first day of disease. On the seventh day after AMI only the CP of MN cells had increased significantly in patients with AMI, while percentage phagocytosis, CP and capacity of phagocytosis of PMN cells increased during the acute phase of AMI. CONCLUSIONS: These data suggest that AMI was followed with a strongly systemic inflammatory response to myocardial damage. Furthermore, activated MN and PMN cells may be a significant source of free radicals that may be involved in lipid peroxidation and produce tissue damage in the early postinfarction period.