Enumeration of haemagglutinin-specific CD8+ T cells after influenza vaccination using MHC class I peptide tetramers.

With emergence of MHC class I tetramers loaded with CD8+ T-cell viral epitopes, it is possible to study virus-specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)-specific T cells in 26 healthy influenza-vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four-colour flow cytometry was used for monitoring of vaccine induced T-cell response. In 15 donors, two- to fivefold increase in frequency of HA-specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA-specific CD8+/IFN-gamma+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA-specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA-specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection.

Outcome of influenza vaccination in combat-related post-traumatic stress disorder (PTSD) patients.

Post-traumatic stress disorder (PTSD) is an anxiety disorder that can occur after exposure to extreme traumatic experience such as war trauma, and is accompanied by fear, helplessness or horror. Exposure to trauma can result in immune dysregulation and influence susceptibility to infectious disease as well as vaccine efficacy. The aim of the study was to determine the relation of psychological stress and the immune response to influenza vaccination in combat-related PTSD patients (n = 28). Detection of anti-viral antibody titre was performed by inhibition of haemagglutination assay. Ex vivo tetramer staining of CD8(+) T lymphocytes was used to monitor T cells specific for human leucocyte antigen (HLA)-A*0201-restricted influenza A haemagglutinin antigens before and after vaccination. Twenty patients showed a fourfold antibody titre increase to one or both influenza A viral strains, and 18 of them showed the same response for both influenza B viral strains. Ten of 15 healthy controls showed a fourfold rise in antibody titre to both influenza A viral strains and eight of them showed the same response for both influenza B viral strains. HLA-A*0201(+) PTSD patients (n = 10) showed a significant increase of influenza-specific CD8 T cells after vaccination. Although those PTSD patients had a lower number of influenza-specific CD8(+) T cells before vaccination compared to HLA-A*0201(+) healthy controls (n = 6), there was no difference in influenza A antibody titre between PTSD patients and control subjects before vaccination. The generated humoral and cellular immune response in PTSD patients argues against the hypothesis that combat-related PTSD in war veterans might affect protection following influenza vaccination.

Chemokines CXCL10 and CXCL11 in the cerebrospinal fluid of patients with tick-borne encephalitis.

OBJECTIVES: The aim of our study was to determine whether cerebrospinal fluid (CSF) of patients with tick-borne encephalitis (TBE) contains CXCL10, CXCL11, p40 subunit of interleukin-12 (IL-12)/IL-23, IL-18 and IL-15. We compared serum and CSF concentrations of CXCL10 and analysed the possible concentration gradient of this chemokine between the periphery and central nervous system. MATERIALS AND METHODS: The study enrolled 19 TBE patients and 10 patients with non-inflammatory neurological diseases. RESULTS: CSF of TBE patients contained CXCL10 (median 217 pg/ml), CXCL11 (8.3 pg/ml), p40 subunit of IL-12/IL-23 (38.9 pg/ml), IL-18 (30.1 pg/ml) and IL-15 (5.9 pg/ml). CXCL10 in the CSF of TBE patients was higher compared with serum (median 62 pg/ml, P < 0.001). CONCLUSION: CSF of TBE patients contains CXCL10, CXCL11, p40 subunit of IL-12/IL-23, IL-18 and IL-15. Increased CXCL10 concentration in CSF suggests a role for this chemokine in the recruitment of CXCR3-expressing T-cells into the CSF of TBE patients.

Establishment of an active laboratory-based surveillance for bacterial meningitis in Croatia and molecular characterization of Neisseria meningitidis isolates causing meningococcal disease that were collected in the year 2000, the first year of activity.

In 2000, 23 Neisseria meningitidis (meningococcal [Men]) isolates were collected in Croatia through an active laboratory-based surveillance for bacterial meningitis (17 Men serogroup B [MenB], 4 MenC, 1 MenW135, and 1 nongroupable isolate). Molecular characterization revealed a substantial level of diversity with only six isolates belonging to electrophoretic type 5 (ET-5) and ET-37 hypervirulent complexes.

Increased numbers of CD38 molecules on bright CD8+ T lymphocytes in infectious mononucleosis caused by Epstein-Barr virus infection.

The aim of this study was to quantify the expression of CD38 on CD8+ T lymphocytes of patients with infectious mononucleosis (IM) caused by Epstein-Barr virus (EBV) and cytomegalovirus (CMV). CD38 quantification technique chosen for this study was based on the enumeration of CD38 antibody binding sites in comparison to the quantification standards rather than determining relative fluorescence, which is difficult to standardize. The study enrolled 19 patients with typical clinical and laboratory parameters compatible with EBV-induced IM as well as 10 patients with atypical clinical presentation of this disease. Furthermore, CD38 expression was analysed in a group of 13 patients with IM caused by CMV infection. CD38 quantification was performed within 6 days of the presentation of symptoms. All three groups of IM patients showed a statistically significant increase in the number of anti-CD38 antibody binding sites (which correspond to the number of CD38 molecules) on bright CD8+ T lymphocytes compared to healthy controls. The numbers of CD38 molecules expressed on CD8+ T lymphocytes did not differ significantly between IM patients with typical and atypical clinical presentation of the disease. Patients with CMV-induced IM had significantly lower numbers of CD38 molecules expressed on CD8+ T lymphocytes. Therefore, we conclude that CD38 quantification could be helpful in differential diagnostics of IM cases with atypical clinical presentation.

AIDS-related body cavity-based lymphoma. A case report.

BACKGROUND: Body cavity-based lymphomas are rare malignancies in human immunodeficiency virus (HIV)-infected patients, but because of their unusual clinical, morphologic and immunophenotypic features, they are recognized as a distinct subgroup of lymphomas connected to human herpesvirus 8 (HHV-8) infection. CASE: A 39-year-old, HIV-positive, homosexual man was admitted to the hospital because of a left-sided pleural effusion that contained malignant lymphoid cells. He responded partially to a low-dose cyclophosphamide/doxorubycin/vincristine/prednisone regimen and died five months after the diagnosis of lymphoma. On cytology, the sediments contained exclusively large, round, neoplastic, lymphoid cells with abundant basophilic cytoplasm and large, round nuclei with prominent nucleoli. Many cells had immunoblastic features, and some had plasmocytoid differentiation. Mitotic figures were numerous. On flow cytometry, the homogeneous population of large cells expressed CD45, CD38, HLA-DR and CD7 positivity. Other specific T-, B- and NK-cell markers tested negative. Polymerase chain reaction demonstrated Epstein-Barr virus (EBV) and HHV-8 in the malignant effusion. CONCLUSION: Primary effusion from lymphoma with molecular evidence of HHV-8 and EBV coinfection represents a distinct clinical and morphologic entity in AIDS patients. However, immunophenotypic markers of malignant clones can be diverse in different cases.

Secondary hyperparathyroidism and brown tumor in dialyzed patients.

Secondary hyperparathyroidism is one of the most common complications of chronic renal failure (CRF). Its pathogenesis is multifactorial and still not completely understood. Pathological mechanism of hypocalcemia, hyperphosphatemia and calcitriol deficiency are basic characteristics of CRF and main reason for morphological changes in parathyroid glands and hyperparathyroidism (HP). We present a case of a female patient born in 1975. At the age of 10, a urinary infection was diagnosed for the first time and treated. Six years later, as nausea and vomiting started, CRF based on bilateral reflux was diagnosed and the patient was included in the hemodialysis treatment. The patient was again examined in 1997, when biochemical parameters, including the level of parathyroid hormone, ultrasonography of the neck, scintigraphy of the skeleton and densitometry revealed secondary HP. Parathyreoidectomy was perfomed in 1998. During the follow up period, a tumefaction on a ramus mandibulae dex. was noticed, which was cytologically diagnosed as osteitis fibrosa, "brown tumor", a rare complication of the secondary HP. Surgery was performed and PHD was granuloma gigantocelulare. Prevention and therapy of secondary HP is a problem that demands early actions to avoid possible complications.

Molecular detection of human papillomavirus in women with minor-grade cervical cytology abnormalities.

BACKGROUND: Human papillomavirus (HPV) has been shown to be the major risk factor for the development of cervical carcinoma, the second most common cancer among women worldwide. Cervical cytology has been the main screening tool for detection of premalignant lesions in last 50 years. OBJECTIVE: The utility of a molecular assay for detection of HPV in cervical smears was evaluated. STUDY DESIGN: A total of 466 women with minor-grade cervical cytology abnormality supposed to be produced by HPV were included. Patients were classified into three groups: Patients with reactive changes, patients with cervical intraepithelial neoplasia grade 1 (CIN 1), and patients with cervical intraepithelial neoplasia grade 2 (CIN 2). In all patients, another cervical swab was obtained and tested for the HPV genome using the Digene Hybrid Capture II. This assay is able to distinguish between high-risk and low-risk HPV types. RESULTS: Based on cytology results, 44 patients showed reactive changes, 250 patients displayed CIN 1, and 172 patients displayed CIN 2. With the molecular assay, HPV was detected in 289/466 (62%) patients. The high-risk HPV type was present in 263 (56.4%) patients and the low-risk type in 26 (5,5%) patients. In 25% of patients with reactive changes, the HPV genome was detected. Corresponding rates for patients with CIN 1 and CIN 2 were 55 and 81%, respectively. CONCLUSION: Molecular detection of HPV should additionally be used to cytology in patients whose cervical smears display reactive changes, CIN 1, or CIN 2. The employed assay allows identification of patients who are at risk for development of high-grade cervical lesions.

[Herpes simplex viruses: biological characteristics, immunopathogenesis, diagnosis and treatment]. / Herpes simpleks-virusi: bioloske osobine, imunopatogeneza, dijagnostika i lijecenje.

Herpes simplex virus (HSV) is the most common cause of sporadic encephalitis in developed countries. Herpes simplex virus type 1 (HSV-1) causes oropharyngeal infections, keratoconjunctivitis and infections of the central nervous system, while herpes simplex virus type 2 (HSV-2) in the immunocompetent most frequently causes genital infections. HSV-1 primary infection usually occurs in the early childhood but is also possible at adolescent age. HSV-2 primary infection is usually postponed till the adult age and coincides with the sexual activity. Common characteristics of these two viruses are a relatively rapid reproductive cycle, an efficient elimination of the infected cells, and the ability of causing a latent infection in the sensory ganglia. Since nowadays there is a specific therapy, the prognoses of severe HSV infections are much better. However, it is necessary that the antiviral therapy be applied shortly after the first symptoms of the disease have appeared. Therefore, the application of a rapid and safe method for detection of HSV from clinical materials is the first step in the treatment of severe and lethal infections like meningoencephalitis. In that light, the method called polymerase chain reaction (PCR) represents a new in vitro technique of DNA replication which enables exponential replication of a well defined DNA fragment. The advantages of this diagnostic method are its rapidity and sensitivity, and it does not require live cells for virus detection.

Immune, endocrine, and psychological responses in civilians displaced by war.

OBJECTIVES: The objectives of this study were to assess the influence of trauma caused by forced expulsion from home in a war-ravaged region on the psychological, hormonal, and immune responses in displaced persons and to analyze the relationships between psychometric, hormonal, and immunologic variables. METHODS: Participants were 20 displaced and 14 control women. Psychosomatic response was evaluated using the COR-NEX2 test. Serum concentrations of cortisol, prolactin, endorphin, thyroxine, and triiodothyronine were measured by radioimmunoassay. Immunophenotyping and lymphocyte proliferation were determined by flow cytometry, and phagocyte functions (i.e., ingestion and antibody-dependent cytotoxicity) against 51Cr-labeled sheep red blood cells were assessed through radioactivity uptake and release, respectively. RESULTS: In comparison with control women, displaced women had higher COR-NEX2 test scores; higher serum cortisol, prolactin, and endorphin levels; an increase in activated phenotype within all three measured cell populations (i.e., B, T, and natural killer cells); as well as an enhanced proportion of proliferating lymphocytes in freshly isolated samples. However, the phytohemagglutinin-stimulated proliferative response, estimated as the stimulation index, was lower in displaced women. A complex pattern of relations between psychological, hormonal, and immune responses was observed. CONCLUSIONS: Chronic psychological stress elicited multiple, predominantly stimulatory influences on immune functions.

Morphologic and functional changes of left ventricle in dialyzed patients after treatment with recombinant human erythropoietin (r-HuEPO).

Dysfunction of the cardiovascular system is a common complication of chronic renal insufficiency. Many factors can cause left ventricular hypertrophy (LVH), and hypertension and anemia are among them. They play an important role in the pathogenesis of LVH as well as in the development of cardiac dysfunction. Echocardiography enables early detection of functional macrocirculatory changes as well as adequate measuring of cardiac structures and LV mass. Anemia of end-stage chronic renal insufficiency (ESRD) is only one among its many complications and has complex pathogenesis; one of the primary factors causing anemia is insufficient production of erythropoietin, a leading factor in the production of erythropoiesis. Anemia correction with recombinant human erythropoietin (r-HuEPO) in ESRD has a positive effect on the cardiovascular system. In this study the authors examined the hemodynamic effect of erythropoietin in anemic patients undergoing hemodialysis and observed its positive effect on the cardiovascular system. Twenty-two patients were included in the study (13 men and 9 women) mean age x=39.5 years. All patients were dialyzed three times a week for 4 hours and were all (Abstract continued) treated, according to protocol, with r-HuEPO for 8 months. Left ventricular mass was measured by the Penn Convention formula. The authors noticed the effectiveness of this therapy through an increase of hemoglobin of 35% and of hematocrit of 34% and a direct effect on the cardiovascular system. Echocardiographic findings showed decrease of LV mass from 391 to 274 mg (30%). The correction of renal anemia with erythropoietin leads to structural microcirculatory changes and partial morphologic regression of preexistent LVH, which again leads to regression of cardiac dysfunctions and improved hemodynamic effect, physical capacity, and cardiopulmonary status, and ultimately better quality of life for dialyzed patients.

HCV genotypes in patients with chronic hepatitis C in Croatia.

The determination of hepatitis C virus (HCV) genotype is an important epidemiological parameter in patients with chronic hepatitis C, while its clinical significance is still a matter of debate. The HCV genotypes in a group of 203 Croatian patients with chronic hepatitis C were examined. Genotypes were correlated to different risk factors, age, duration of the disease, liver histology activity and level of viremia. HCV-RNA was detected in each serum by means of reverse transcription PCR. Genotypes were determined from the amplificate by reverse hybridization in a line probe assay. The level of viremia was assessed by branched DNA (bDNA) signal amplification. The most common genotype was 1b (61.1% of patients), followed by 3a (26.1%), and 1a (10.8%). Other genotypes such as 2a and 4 were only rarely found in our patients (2%). Genotype 1b was most commonly acquired via blood transfusion, while genotype 3a was strongly related to intravenous drug use. Genotype 1b was associated with older age (mean 42.6 vs 29.3 years), longer duration of the disease (mean 6.0 vs 3.5 years), higher histologic activity score (mean 13.2 vs 10.6) and higher viremia (mean 9.06 vs 5.93 Meq/ml) at statistically significant levels (p < 0.001) when compared to genotype 3a. The prevalence of HCV genotypes follows the patterns of southeastern European countries, except for a lower prevalence of genotype 2. The observation that genotype 1b is associated with higher viremia and more severe liver injury is confirmed.

Cytologic and immunoenzymatic findings in CSF from patients with tick-borne encephalitis.

OBJECTIVE: To evaluate the dynamics of cytologic changes in the cerebrospinal fluid (CSF) of patients with tick-borne encephalitis (TBE) and to determine the various forms of mononuclear cells by immunocytochemical methods. STUDY DESIGN: To perform a cytologic analysis of 200 cells in stained sediment of CSF. To determine the population and subpopulation of mononuclear cells by using monoclonal antibodies. RESULTS: Pleocytosis in CSF in patients with TBE during the first three days of illness was, on average, 570 cells per cubic millimeter with about 60% neutrophils. Mononuclear cells predominated from the fifth day of illness. Among lymphoid cells, 18% carried marker CD20 and 60% of them marker CD3. Among monocytoid cells, 66% carried marker CD32. CONCLUSION: CSF cytology findings reflect the immunologic and inflammatory changes in the CNS during TBE.

Comparison of lymphocyte subpopulations in the peripheral blood of patients with infectious mononucleosis and human immunodeficiency virus infection: a preliminary report.

The aim of this study was to analyse the lymphocyte subpopulations in the peripheral blood of HIV-1-infected adults to compare them with four patients with acute EBV infection. Lymphocyte subsets in 15 healthy controls, 40 HIV-1-infected adults and 4 EBV-infected patients with infectious mononucleosis were analysed by flow cytometry. The immunophenotyping of HIV-1-infected patients in different stages of disease showed a significant reduction in the percentage and absolute count of CD4+ T-lymphocytes, significantly increased percentage of CD8+ T-lymphocytes, inverted CD4/CD8 ratio and an increase in the expression of activation marker HLA-DR compared to controls. The immuno-phenotyping profiles of HIV and EBV infection share some similarities as they both result in the decreased percentage of CD4+ T-lymphocytes, increased CD8+ T-lymphocytes and an inverted CD4/CD8 ratio. Patients with HIV infection could be distinguished from patients with EBV infection by the absolute lymphocytosis and increased expression of HLA-DR seen in the patients with infectious mononucleosis. In conclusion, both HIV-1 and EBV profoundly change the distribution of lymphocyte subpopulations in the peripheral blood. It is our opinion that flow cytometry could be an aid in the rapid distinguishing of patients with suspected primary HIV-1 infection from those with infectious mononucleosis (before serology data are available).

A case of McLeod syndrome with chronic renal failure.

A 50-year-old man with the rare McLeod syndrome, associated with glomerular lesion to the end stage of chronic renal failure and death, is reported. McLeod syndrome is an X-linked recessive disorder on the basis of abnormal expression of the Kell blood group antigens and absence of erythrocyte surface Kx antigen. Most often the clinical and pathological findings are retinitis pigmentosa to blindness, progressive chronic neuropathy, cortical atrophy, dilated cardiomyopathy, and glomerular lesion with chronic renal failure. Among the laboratory parameters the most important are very low level of cholesterol and triglycerides, then various numbers of acanthocytes in peripheral blood smears and sometimes in urine (as in our case).

Comparative study of mitral annular calcification (MAC) with cardiac arrhythmias in dialysis patients.

Cardiac arrhythmias and myocardial malfunction are very frequent in uremic patients. The pathogenesis and etiology of arrhythmias are very complex and still unknown. The sedimentation of calcium salt in myocardial structures is one of the reasons for emergence of cardiac arrhythmias (AV conduction defects, ectopic arrhythmias). The appearance of mitral annular calcification (MAC), as the expression of the speed up process of atherosclerosis, was noted in younger uremic patients especially during hemodialysis. The aim of our research was to compare the incidence of MAC and cardiac arrhythmias in patients on hemodialysis. Our study included 40 patients, 24 male and 16 female, in the age between 20 and 60. Patients were mostly from Zagreb and the Counties of Zagreb (35%), Karlovac (10%), Slavonski Brod (7.5%), Varazdin (5%) and Pozega (5%). All 40 patients received 24 hours of Holter monitoring and 2-D echocardiography of M-mode. The patients were divided in two groups: I MAC+ (N = 23) and II MAC- (N = 17). Frequency of cardiac arrhythmias in group I was: atrial fibrillation N = 0; conduction defects N = 2 (1%); ventricularectopy Lown grade 3-5 N = 15 (65%); supraventricular ectopy N = 8 (34%), while the frequency of cardiac arrhythmias in group II was: atrial fibrillation N = 0; conduction defects N = 0; ventricular ectopy Lown grade 3-5 N = 6 (35%), supraventricular ectopy N = 6 (35%). During statistical processing the significant connection of MAC+ and frequency of cardiac arrhythmias was noticed. For both groups we have not noticed statistical significance in cardiac arrhythmia compared to electrolytes, risk factors PTH, and age. The time of hemodialysis treatment is one of possible factors for incidence of cardiac arrhythmias influenced by MAC. We noticed statistically significant (p < 0.05) difference of rhythm disorders between group I and group II especially for the ventricular ectopic activity, the frequency of which was higher in group I than in group II. MAC has probably significant role in dialysis patients for the development of cardiac arrhythmias within the framework of series of complicated multifactorial patogenetic mechanisms.

[Kawasaki disease--case reports of 2 female patients]. / Kawasakieva bolest--prikaz dviju bolesnica.

Clinical and laboratory features of two female patients with Kawasaki disease, aged 9 and 12 years are presented. The main pathogenetic, differential diagnostic and therapeutic considerations are discussed.

Morphological and cytochemical characteristics of atypical mononuclear cells in the blood of patients with the syndrome of infectious mononucleosis caused by Epstein-Barr virus.

On the basis of their origin and morphologic and cytochemical characteristics, atypical mononuclear cells (AMNC) in the peripheral blood of patients with the syndrome of infectious mononucleosis caused by the Epstein-Barr (EB) virus can be classified into three groups, from I to III. The AMNC groups I are the most numerous (76.36% +/- 2.50%). A larger part of these cells have the characteristics of more or less altered lymphoblasts and the smaller part of large pyroninophilic cells and "reactive immunoblasts", and meant to have developed from T lymphocytes. These are the most numerous in the acute phase of the disease, which correlates with the most significant increase of T lymphocytes values. A very high percentage (84.00% +/- 7.30%) of the AMNC group I contains acid phosphatase isoenzyme 3 and acid non-specific esterase (82.43% +/- 7.76%), a considerable percentage of beta-glucuronidase (58.75% +/- 13.99%), and acid phosphatase isoenzyme 1 (51.20% +/- 10.81%). A small percentage of these cells contains acid phosphatase isoenzyme 5 resistant to the action of L+tartaric acid. In 39.04% +/- 8.72% of the lymphocytes in the peripheral blood of the examined patients and in the two thirds and nearly three fourths of the AMNC group I, the presence of acid phosphatase manifests itself in the form of scattered granules. The positive reaction to non-specific esterase (pH 7.8-8) in 39.72% +/- 8.44% lymphocytes of the peripheral blood of the patients and in 59.26% +/- 10.09% of the AMNC group I manifests itself in the form of scattered granules.(ABSTRACT TRUNCATED AT 250 WORDS)