Bioluminescence Imaging of Adoptively Transferred Lymphocytes During Allogeneic Tumor Rejection.

AIM: The aim of the present study was to analyze the survival, spatial distribution and proliferation of adoptively transferred lymphocytes in allogeneic tumor rejection. MATERIALS AND METHODS: Transgenic ß-actin-luc mice that express luciferase were sensitized against SL2 tumors and were used as lymphocyte donors to study the anti-tumor effect in SL2 tumor-bearing lymphocyte-deficient RAG(-/-) mice. Whole-body bioluminescence images of recipient mice were obtained to track the adoptively transferred lymphocytes. Proliferation of lymphocytes was estimated by quantification of photon emission. RESULTS: T lymphocytes sensitized against allogeneic SL2 tumors cured the majority of SL2 tumor-bearing RAG(-/-) mice. Bioluminescence imaging showed that transferred T lymphocytes survived in the spleen and lymph nodes. Tumor rejection was associated with lymphocyte proliferation and migration to the tumor site. CONCLUSION: Sensitized T lymphocytes from transgenic ß-actin-luc mice reject allogeneic SL2 tumors in RAG(-/-) mice and can be tracked in vivo using bioluminescence imaging.

Nevomelanocytic atypia detection by in vivo reflectance confocal microscopy.

BACKGROUND AND OBJECTIVE: In vivo reflectance confocal microscopy (RCM) is a promising novel technology for non-invasive early diagnostics of cutaneous melanoma. However, the possibility to detect melanocytic atypia in nevi by means of in vivo RCM remains unknown. The aim of the study was to evaluate the significance of in vivo RCM features of melanocytic atypia for the diagnosis of melanocytic nevi, dysplastic nevi and cutaneous melanoma. MATERIALS AND METHODS: A total of 138 melanocytic skin lesions comprising 25 melanocytic nevi, 69 dysplastic nevi and 44 melanomas were analyzed by means of dermoscopy, in vivo RCM and routine histopathology. In vivo RCM images were analyzed for the arrangement of keratinocytes in epidermis, pagetoid cells and junctional melanocytic nests and correlated refractivity aspects of nests with histopathology. RESULTS: Separately and all together taken the in vivo RCM features of melanocytic atypia were significant in differential diagnosis of benign and malignant melanocytic skin lesions, though none of the features was significant in discriminating nevi without cytologic atypia of dysplastic nevi. In vivo RCM feature of dense cell clusters corresponded with melanin containing nevomelanocytes on histopathology though exact correspondence of non-homogeneous and atypical sparse cell clusters remained questionable. CONCLUSIONS: Nevus with histopathologically confirmed nevomelanocytic atypia (dysplastic nevus) could not be distinguished from nevus without atypia using analyzed in vivo RCM features of melanocytic atypia. More accurate diagnostics by means of in vivo RCM needs further investigation on reflectance of single and nested cutaneous melanocytes in benign and malignant skin lesions.

Role of marker lesion when applying intravesical instillations of IL-2 for non-muscle-invasive bladder cancer comparison of the therapeutic effects in two pilot studies.

AIM: Comparison of the therapeutic effect of treatment of non-muscle invasive bladder carcinoma (NMIBC) after intravesical Interleukin-2 (IL-2) instillations in the presence and absence of a marker tumour. MATERIALS AND METHODS: Two pilot studies were performed in patients with NMIBC. The first study (10 patients) was performed in Krakow (Poland), the second (26 patients) in Vilnius (Lithuania). In Krakow the tumours were treated with incomplete transurethral resection (TUR) leaving a marker tumour of 0.5-1.0-cm followed by IL-2 instillations (3 × 10(6) IU IL-2) on five consecutive days. In Vilnius the tumours were treated with complete TUR, followed by IL-2 instillations (9 × 10(6) IU IL-2) on five consecutive days. RESULTS: During 30 months follow-up, the recurrence-free survival was 5/10 (50%) and 6/26 (23%) after incomplete and complete TUR, respectively. So, the ratio of the recurrence-free survival after incomplete/complete TUR of 50/23=2.2. The median of the recurrence-free survival is >20.5 months and 7 months after incomplete and complete TUR, respectively. So, this ratio was >20.5/7= >2.9. The hazard ratio which combines both the chance of the disease recurrence and its timing for both censored and uncensored cases was 0.53, again confirming the better outcome after incomplete TUR. CONCLUSION: A possible explanation for the better therapeutic effects after incomplete TUR compared with complete TUR is that the marker tumour has tumour-associated antigens (TAA) that could lead to an immune reaction that is stimulated by local application of IL-2. After complete TUR, no TAA are available to initiate and to stimulate an immune reaction; consequently, local IL-2 therapy is less effective after complete TUR. The results of these two pilot studies have led to the recent start of a randomised prospective clinical trial in which therapeutic effects of local IL-2 therapy after complete and incomplete TUR are compared.

Expanded effector memory T-lymphocytes in DBA/2 mice do not inhibit the growth of SL2 tumours.

BACKGROUND: The aim of this study was to analyse changes in levels of memory T-lymphocytes during growth of SL2 tumours in DBA/2 mice and to evaluate whether these lymphocytes may have an inhibitory effect on tumour growth. MATERIALS AND METHODS: Percentages of naïve (CD8+CD44lowCD62L+), central memory (CD8+CD44high CD62L+) and effector memory (CD8+CD44highCD62L-) lymphocytes in the CD8+ subset in peripheral blood, spleen and lymph nodes of tumour-bearing and control mice were analysed by flow cytometry. RESULTS: The percentage of effector memory lymphocytes in the CD8+ subset increased during growth of tumours, whereas that of naïve CD8+ lymphocytes decreased. No correlation between the levels of effector memory lymphocytes in peripheral blood and the mass of tumours was found. CONCLUSION: SL2 tumours induce expansion of effector memory lymphocytes in DBA/2 mice. However, expanded effector memory lymphocytes do not inhibit the growth of tumours.

Prognostic significance of peripheral blood CD8highCD57+ lymphocytes in bladder carcinoma patients after intravesical IL-2.

BACKGROUND: The objective of this study was to evaluate the recurrence-preventing effect of intravesical instillations of interleukin-2 (IL-2) in patients with non-muscle-invasive bladder carcinoma. In addition, this study aimed to determine the significance of immune parameters for recurrence-free interval. PATIENTS AND METHODS: Twenty-six patients with non-muscle-invasive bladder carcinoma were treated with intravesical instillations of IL-2 (Proleukin®, Novartis, formerly Chiron) in doses of 9 × 10(6) IU on 5 consecutive days, beginning on the second day after transurethral resection (TUR) of tumours. CD8(high)CD57(+) lymphocytes in peripheral blood were determined before TUR and compared with the recurrence-free interval after treatment. RESULTS: The multivariate analysis showed that CD8(high)CD57(+) lymphocytes had a prognostic significance in combination with number of bladder tumours, prior recurrence rate and age of patients. CONCLUSION: Peripheral blood CD8(high)CD57(+) lymphocytes have prognostic significance for recurrence-free survival in patients with non-muscle-invasive bladder carcinoma after TUR and intravesical IL-2.

Aetiological diagnostics of acute bacterial meningitis in children.

Aetiology of bacterial meningitis (BM) can be confirmed by various microbiological methods. The aim of this study was to assess the role of microbiological methods used for confirmation of BM in children and determine the influence of the aetiological agent, patient age and antibacterial treatment on study results. Over a 5-y period (1998-2002) BM was diagnosed in 90 children at Vilnius University Centre for Paediatrics. Aetiology was confirmed by cerebrospinal fluid (CSF) and blood culture, microscopic CSF smear examination, CSF and blood latex agglutination test. CSF and blood cultures were positive in 53% and 39% of cases, respectively. Microscopic CSF smear examination was positive for 57% of the specimens. CSF latex agglutination was positive in 64% and blood in 47% of cases. Causative agent and received antibiotic therapy prior to investigation of obtained material affected some final microbiological results. However, no influence of patient age was found. Microbiological confirmation was achieved in 59% of cases using CSF and/or blood culture and in 78% of cases using all available methods in practice. The most common pathogens of bacterial meningitis were H. influenzae type b, N. meningitidis and S. pneumoniae.

A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample.

BACKGROUND: Presently available flow cytometric methods of bromodeoxyuridine (BrdUrd) labelling do not provide information on the cell cycle time (TC) and the growth fraction (GF). In this paper, we describe a novel and simple method to estimate TC and GF from flow cytometric analysis of a single tumour sample after BrdUrd labelling. METHODS: The proposed method is based on two assumptions: (1) the number of labelled cells traversing the cell cycle per unit time is constant and (2) the total number of labelled cells is constant throughout the cycle, provided that cells produced after division are excluded. The total numbers of labelled divided G1 cells, labelled divided S cells, labelled undivided S cells, and labelled undivided G2 cells were obtained for DNA histograms of BrdUrd-positive cells in a collected sample. These cell numbers were used to write equations to determine the durations of cell cycle phases, TC and GF. To illustrate the application of the proposed formulae, cell cycle kinetic parameters were analysed in solid SL2 tumours growing in DBA/2 mice and in human T-leukaemia Jurkat cells in culture. RESULTS: The suitability of the proposed method for estimating durations of the cell cycle phases, TC and GF was demonstrated. TC in SL2 tumours was found to be relatively constant at 4 and 10 days after tumour implantation (20.3 +/- 1.1 h and 21.6 +/- 0.9 h, respectively). GF in tumours at day 10 was lower than GF at day 4 (54.2 +/- 7.7% vs. 79.2 +/- 5.9%, p = 0.0003). Approximate values of TC and GF of cultured Jurkat cells were 23.9 h and 79.3%, respectively. CONCLUSION: The proposed method is relatively simple and permits estimation of the cell cycle parameters, including TC and GF, from a single tumour sample after labelling with BrdUrd. We have shown that this method may be useful in preclinical studies, allowing estimation of changes in GF during growth of murine tumours. Experiments with human Jurkat cells suggest that the proposed method might also prove suitable for measurement of cell kinetics in human tumours. Development of suitable software enabling more objective interpretation of the DNA profile in this method would be desirable.

Clinical presentation of pertussis in fully immunized children in Lithuania.

BACKGROUND: In Lithuania, the vaccination coverage against pertussis is high. Nevertheless, there is a significant increase in pertussis cases in fully immunized children. The aim of our study was to determine the frequency of classical symptoms of laboratory confirmed pertussis and describe its epidemiology in children fully vaccinated against pertussis. METHODS: From May to December 2001, 70 children aged 1 month to 15 years, suffering from prolonged cough were investigated in the Centre of Paediatrics, Vilnius University Children's Hospital. The collected information included personal data, vaccination history, clinical symptoms of the current illness, and treatment before hospitalization. At the admission to the hospital blood samples were taken from all studied children for Bordetella pertussis IgM and IgA. RESULTS: A total of 53 (75.7%) of the 70 recruited patients with prolonged cough showed laboratory evidence of pertussis. 32 of them were fully vaccinated with whole cell pertussis vaccine (DTP). The age of fully vaccinated patients varied from 4 to 15 years (average 10.9 +/- 3.1; median 11). The time period between the last vaccination dose (fourth) and the clinical manifestation of pertussis was 2.6-13 years (average 8.9 +/- 3.0; median 9). More than half of the children before the beginning of pertussis were in contact with persons suffering from long lasting cough illness in the family, school or day-care center. The mean duration from onset of pertussis symptoms until hospitalization was 61.4 +/- 68.3 days (range, 7 to 270 days; median 30). For 11 patients who had had two episodes (waves) of coughing, the median duration of cough was 90 days, and for 21 with one episode 30 days (p < 0.0002). Most of the children (84.4%) had paroxysmal cough, 31.3% had post-tussive vomiting, 28.1% typical whoop, and 3.1% apnea. Only 15.6% children had atypical symptoms of pertussis. CONCLUSION: Fully vaccinated children fell ill with pertussis at the median of 11 years old, 9 years following pertussis vaccination. More than half of the children could catch pertussis at home, at school or day-care center. Clinical picture of pertussis in previously immunized children is usually characterized by such classical symptoms as prolonged and paroxysmal cough, rarely by whopping and post-tussive vomiting, and very rarely by apnea.

[Hypotensive epidural anesthesia in thoracic surgery]. / Hipotenzine epidurine anestezija torakaliniu operaciju metu.

OBJECTIVE: This study evaluated effectiveness of hypotensive epidural anesthesia in decreasing blood loss, operation and extubation time, opiates use and stay in intensive care unit. MATERIAL AND METHODS: Fifty-eight patients were enrolled in the study. Right (n=16) or left (n=42) pneumectomy was performed for the study patients. We used the hypotensive anesthesia induced by thoracic epidural anesthesia for the group T (n=29/50%) and normotensive anesthesia for the group K (n=29/50%). The epidural catheter was introduced into the epidural space at the level Th4-5. Arterial pressure was reduced using bupivacaine into epidural space. In the group T median arterial pressure was about 50-60 mmHg. For the group K we used only general anesthesia and median arterial pressure was 80-120 mmHg. RESULTS: The average intra-operative blood loss was 534+/-198 ml in the group T and 1287+/-380 ml in the group K (p<0.001). Post-operative blood loss was the same in both groups. The average operation time was 10% shorter in the group T (p=0.078). Fentanyl use in the T group was 203+/-91 microg and 1266+/-601 microg in the K group (p<0.001). Patients in the T group were safely extubated after 66+/-17 min (p<0.001) and discharged from intensive care unit after 2+/-1.1 days (p<0.05). The patients in the group K were extubated after 138+/-37 min and discharged from intensive care unit after 3.27+/-1.3 days. CONCLUSION: Hypotensive epidural anesthesia is an effective method to decrease blood loss and blood transfusions in thoracic surgery. It creates better conditions for surgery and reduces stay in intensive care unit. Also there were no serious cardiac, neurological and renal intra-operative and post-operative complications that could be conditioned by the use of hypotension.

Management of Lithuanian children's acute diarrhoea with Gastrolit solution and dioctahedral smectite.

OBJECTIVE: Acute gastroenteritis represents a major cause of morbidity and mortality worldwide among children, and rehydration treatment has been one of the cornerstones in the management strategy. The natural clay dioctahedral smectite (Smecta) increases intestinal barrier function and is effective against infectious diarrhoea in children. The purpose of this work was to compare the efficacy and tolerance of Lithuanian children's diarrhoea treatment with dioctahedral smectite combined with hypotonic oral rehydration solution (ORS)--Gastrolit--versus Gastrolit alone to establish the influence of Smecta on serum electrolyte balance in young children with diarrhoea and mild or moderate dehydration. METHODS: Smecta combined with ORS (study group) and ORS alone (control group) were evaluated in a multicentre, open, randomized trial in 54 children aged 6-48 months hospitalized for acute diarrhoea (mostly rotavirus aetiology) and signs of mild and moderate dehydration. The main outcomes examined were duration of diarrhoea, fever, number of vomiting episodes, and serum electrolyte balance before and after treatment. RESULTS: The mean duration of diarrhoea was significantly shorter in the study group (42.3 +/- 24.7 h) than in the control group (61.8 +/- 33.9 h). No side effects of Smecta were observed. The changes of sodium, potassium, chloride and calcium concentrations after treatment were minimal and in the normal range. CONCLUSIONS: Smecta significantly reduced the duration of diarrhoea, was safe and well tolerated, and had no impact on the adsorption of electrolytes. Smecta could be used together with ORS in children suffering from acute gastroenteritis (without uncontrollable vomiting) with mild and moderate dehydration.