Applicability of the Demirjian, Willems and Haavikko methods in Croatian children.

Age estimation is an inescapable part of every identification process. During growth and development, it is possible to estimate age based on the developmental stages of teeth. The aim of this study was to evaluate three frequently used methods for dental age estimation on a broad sample of Croatian children. The sample comprised 1996 digital, standardized orthopantomograms of children (1121 boys and 875 girls) aged 5 to 16, collected in four major Croatian cities. Age was estimated according to the Demirjian, Willems and Haavikko methods and the accuracy of the estimation was evaluated. The Kappa for intra-examiner agreement was 0.83 for the Haavikko stages and 0.92 for the Demirjian stages. Using the Demirjian method, the average overestimation of age was 0.80 years for boys and 0.84 years for girls. The Willems method overestimated the mean age by 0.41 years in boys and 0.22 years in girls. The Haavikko method underestimated the mean age by 0.60 years in boys and 0.80 years in girls. The Willems method proved to be the most accurate and can be used for dental age estimation among Croatian children. The Demirjian and Haavikko methods showed greater deviation between dental and chronological age and require adaptation when used in the Croatian population.

Transfer of immunity by transfer of bone marrow cells: a requirement for T lymphocytes and sensitivity to cyclophosphamide.

Thymectomized, lethally irradiated mice reconstituted with bone marrow cells (TIR mice) from normal donors succumbed after i.p. challenge with xenogeneic (rat) Yoshida ascites sarcoma (YAS) given 1 month after irradiation and reconstitution. YAS rejection and production of YAS-antibodies was induced in TIR mice by a single i.v. injection of normal syngeneic spleen cells given 1 day after the tumor. Purified splenic T lymphocytes also induced YAS rejection in TIR mice, but splenic B lymphocytes did not affect progressive tumor growth. Tumor was also rejected in TIR mice that had been reconstituted with bone marrow cells from YAS-immune donors. The sera of these TIR mice did not contain tumor antibodies between reconstitution and YAS challenge, but a high YAS-antibody titer was present after YAS challenge and rejection. Immunofluorescence did not reveal any dramatic differences in the spleen and bone marrow content of T and B lymphocytes of TIR mice reconstituted with cells from normal donors and those reconstituted with cells from the YAS-immunized donors. Transfer of YAS-resistance was abolished when the bone marrow cells from immunized donors were treated with Thy 1.2 antiserum and complement, or when bone marrow donors were injected with cyclophosphamide 1 day after immunization. Cyclophosphamide was also shown to induce strong and specific suppression of YAS-antibody production in normal mice.

One year treatment of Barrett's oesophagus with proton pump inhibitors (a multi-center study).

OBJECTIVES: Aim of the study was to investigate the effects of 1-year therapy by different proton pump inhibitors (PPIs) on epithelial tissue and surrounding inflammatory changes in Barrett's oesophagus, in patients who have abandoned invasive therapy. METHODS: A group of 120 patients (sampled in 60-month period, from 61201 upper gastrointestinal endoscopies) who were diagnosed both, endoscopically and pathohistologically with Barrett's oesophagus, and who have abandoned invasive therapeutic approach were enrolled in study. Treatment with different PPIs was initiated and continued for a year. At the end of treatment, patients were reassessed by endoscopy with tissue biopsy and pathohistological analysis. RESULTS: No difference in regenerating squamous epithelium or degree of dysplasia was seen between different treatment groups. Interestingly, most patients receiving pantoprazole (94%) ended up with thinner squamous epithel (P<0.0001). The squamous epithel was consider thinner only if its total thickness, measured on histological specimen, was smaller for more than 50% of the thickness before therapy. Significantly less of difference (P<0.0014) was seen with patients receiving lansoprazole (65%) and (P<0.003) omeprazole (50%). CONCLUSION: Regeneration of the squamous epithel was the same for all PPIs but not good enough to stop the progression of the disease.

Analysis of growth of multicellular tumour spheroids by mathematical models.

We wished to determine the applicability of previously proposed deterministic mathematical models to description of growth of multicellular tumour spheroids. The models were placed into three general classes: empirical, functional and structural. From these classes, 17 models were applied systematically to growth curves of multicellular tumour spheroids used as paradigms of prevascular and microregional tumour growth. The spheroid growth curves were determined with uniquely high density of measurements and high precision. The theoretical growth curves obtained from the models were fitted by the weighted least-squares method to the 15 measured growth curves, each corresponding to a different cell line. The classical growth models such as von Bertalanffy, logistic and Gompertz were considered as nested within more general models. Our results demonstrate that most models fitted the data fairly well and that criteria other than statistical had to be used for final selection. The Gompertz, the autostimulation and the simple spheroid models were the most appropriate for spheroid growth in the empirical, functional and structural classes of models, respectively. We also showed that some models (e.g. logistic, von Bertalanffy) were clearly inadequate. Thus, contrary to the widely held belief, the sigmoid character of a three or more parameter growth function is not sufficient for adequate fits.

Suppression of rat tumor colonies in the lung by oxygen at high pressure is a local effect.

The effect of hyperbaric oxygen (HBO) on the growth of anaplastic carcinoma colonies in rat lungs after intravenous tumor cell injection was studied. From the first day after tumor cell injection, the rats were exposed to HBO for 16-21 days, 90 min per day. Oxygen at a pressure of 300 kPa (3.0 ATA) significantly decreased the number of lung tumor colonies and increased the survival of tumor-bearing rats, whereas the application of oxygen at a pressure of 100 kPa had no effect. An oxygen-nitrogen normoxic mixture balanced with nitrogen to 300 kPa (3.0 ATA) did not affect the number of colonies, suggesting that the effect was specific for oxygen and not for the increased pressure itself. A 6-day application of oxygen at a 300 kPa pressure suppressed the growth of lung tumor colonies when applied on days 1-6 and 7-12 after intravenous tumor cell injection, but had no effect when applied on days 13-18. In contrast to dramatic effects of HBO on the development of artificial lung metastases, the oxygen at the same 300 kPa pressure had no effect on the growth of tumor cells injected in the hind foot. Thus it appears that the suppression of lung tumor colonies by HBO was due to local oxygen effects in the lungs.

Correlation of morphological FAB classification and immunophenotyping: value in recognition of morphological, cytochemical and immunological characteristics of mixed leukaemias.

Correlation between the FAB classification and immunophenotype was studied in 169 consecutive adult patients with acute leukaemia (AL). The lineage of leukaemic cells could be determined in the majority of cases, whereas 3 patients (1.8%) remained unclassified. In 22 out of 71 patients (31%) with acute myeloid leukaemia (AML) FAB M1 and M2 types, and in 5 out of 16 patients (31%) with chronic myeloid leukaemia (CML) in myeloid blast crisis, leukaemic cells did not express myeloid lineage-related markers, indicating asynchronous expression of cell markers in a substantial proportion of patients. Flow cytometric two-colour immunofluorescence revealed mixed AL immunophenotype in 6 out of 169 patients (3.4%). This group included five CD2+AML (5% of AML tested) and one undifferentiated AL expressing CD10(CALLA), CDw65(VIM-2). The former group included FAB M1, M2, M3 and M4 forms of AML with a single cell population, and an AML M2 patient with both cytochemically and immunologically two separate populations of leukaemic cells. This further illustrates the heterogeneity of the target cell(s) for leukaemogenesis and the level of differentiation of AML cells. However, there was no difference in the treatment response and the remission duration between AML patients and patients with mixed phenotype AML.

Survival analysis of untreated patients with non-small-cell lung cancer.

The survival rate analysis of 130 patients with non-small-cell lung cancer who did not receive any specific anticancer therapy showed no statistically significant differences in the survival rates between various TNM combinations classified into stage groups II, IIIa, IIIb, and IV, as proposed by Mountain in 1989 and adopted by the American Joint Committee on Cancer. Following these findings, based on survival probabilities, two distinctive staging groups could be distinguished. The first stage group was composed of only the T1, 2N0, M0 combination, and the second of all other TNM combinations. In a purely biologic sense of tumor growth, the lymph node involvement appeared to be the crucial factor determining the length of survival.

Pre-treatment of transplant bone marrow cells with hydrocortisone and cyclosporin A alleviates graft-versus-host reaction in a murine allogeneic host-donor combination.

The aim of the study was to alleviate graft-versus-host reaction (GVHR) by pre-treatment of the bone marrow (BM) transplant with hydrocortisone (HC) and cyclosporin A (CsA) in C57BL/6J (donor) --> CBA/J (recipient) mouse combination. BM cells were exposed to HC and CsA for 1 h at 37 degrees C and then injected into lethally irradiated (9.5 Gy) mice at a dose of 2 x 10(6) BM cells/mouse. Haematopoietic recovery was assessed on day 12, and survival was followed for 100 days. Combinations of 1000 microg/ml HC and 100 microg/ml CsA, and 100 microg/ml HC and 10 microg/ml CsA significantly reduced MLR and additively mitigated GVHR in vivo, achieving 40% and 26% survival rates, respectively. However, HC and CsA altered neither the peripheral blood cell counts nor in vitro and in vivo BM cell clonogenic potential. Additional studies have shown that HC and CsA blocked con A-driven differentiation of CD8+ and CD4+ CD8+ lymph node cells (LNC) and progression of LNC to S + G2/M cell cycle phases, and inhibited IL-1, IL-2 and TGF-beta while enhancing GM-CSF gene expression in BM cells. Taken together, these data indicate that the pre-treatment of the BM transplant with HC and CsA results in inactivation of GVHR effector cells and mitigation of GVHR while sparing BM repopulating capacity.

Relationship between differing volumes of bone marrow aspirates and their cellular composition.

We compared the cellular composition of the first 1.0 ml volume bone marrow aspirate with that of an aliquot from the total bone marrow harvest at the end of the procedure in 17 healthy bone marrow donors. Each sample was assayed for its content of red blood cells, nucleated cells, CD2+, CD4+, CD8+, CD19+, HLA-DR+, CD56+, CD13+, CD33+, CD34+ and KiM8+ cells and CFU-GM. On the basis of data obtained, we estimated that the first 1.0 ml samples had 8.0 +/- 5.2% (SD) and the transplant samples 20.8 +/- 8.5% contamination with nucleated blood cells. The calculation revealed that both types of bone marrow samples had 100% volume contamination with peripheral blood, i.e. that bone marrow cells were aspirated within blood fluid volume. Nucleated cell concentration was 3-fold, and CFU-GM concentration 10-fold lower in the transplant than in the first-puncture 1.0 ml bone marrow samples. Various marker-positive cells appeared in transplant samples in concentrations that depended on their abundance in the first-puncture 1.0 ml and blood samples. Taken together, our data suggest that bone marrow harvesting would be substantially improved if individual aspirates were small in volume and taken from bone puncture sites as distant as possible.

Lymphocyte subsets in normal human bone marrow harvested for routine clinical transplantation.

Bone marrow and peripheral blood of 25 healthy bone marrow donors from our allogeneic bone marrow transplantation program were assessed for cell subsets bearing T11(CD2), T4(CD4), T8(CD8), B1(CD20) J5(CALLA, CD10), Mo1(CD11b), MY7(CD13). Mo2(CD14), MY9(CD33) and NKH-1 antigens. Bone marrow cell samples were taken for analysis at the start or at the end of the harvesting procedure of aspiration from the iliac crest. All samples were analysed on a flow cytometer at the lymphocyte window as obtained on the two-parameter (L90oLSxFALS) scatter diagram. There were no differences in the lymphocyte subset composition of bone marrow samples taken at the start or at the end of the harvesting procedure. In contrast to the majority of literature data, a high CD4/CD8 ratio was detected in bone marrow samples: it did not differ from that in the peripheral blood. The proportions of CD2 and CD4 T cell markers in the bone marrow correlated with those in the peripheral blood, thus further documenting a substantial bone marrow contamination with peripheral blood cells. A relatively large aspirate volume (4-5 ml) obtained from individual aspiration sites was identified as the only factor possibly accounting for the high-level contamination of bone marrow samples with peripheral blood. This conclusion was corroborated by low T cell proportions and low CD4/CD8 ratios found in the bone marrow washed from bone fragments and in bone marrow samples aspirated at first bone puncture in a volume of 1.0 ml. Taken together, these findings imply that less vigorous suction may decrease the number of T lymphocytes in bone marrow harvested for transplantation purposes.

Combination of cyclosporin and methotrexate for prophylaxis of acute graft-versus-host disease after allogeneic bone marrow transplantation for leukemia.

From May 1985 to July 1989, 76 patients with leukemia (30 acute myelogenous leukemia, 24 acute lymphoblastic leukemia and 22 chronic myeloid leukemia) were randomized to receive either cyclosporin (CSP) alone (n = 39) or CSP combined with methotrexate (CSP + MTX, n = 37) for graft-versus-host disease (GVHD) prophylaxis. Patients were conditioned with total body radiation and cyclophosphamide followed by bone marrow infusion from an HLA-identical sibling. Engraftment of the transplanted bone marrow was similar in both groups. The incidence of moderate to severe acute GVHD was significantly higher in the CSP group compared with the CSP + MTX group (20 (51%) versus 9 (25%), chi 2 = 4.76, p less than 0.02). There was no significant difference in the incidence of chronic GVHD. Survival was significantly better for the CSP + MTX group (63 +/- 16%) compared to CSP alone (42 +/- 18%). Leukemia-free survival tended to be better for the CSP + MTX group (55 +/- 17% versus 32 +/- 16%).

AgNORs predictive value of prognosis in non-Hodgkin's lymphoma: comparison with flow cytometric cell cycle analysis.

Paraffin-embedded histopathologic specimens, taken before the commencement of therapy from 14 low-grade and 21 high-grade malignant lymphoma patients, and 9 normal lymph nodes were utilized to analyze six cell DNA-related parameters. The flow cytometry technique was used to determine cell-cycle G0/G1, S and G2/M phases, and silver staining to enumerate nuclear organized regions (AgNORs); nucleus surface area was determined by an image-analyzing system. The six parameters and natural logarithm of cell proportion in the S-phase (LS) were determined according to the histologic tumor type and achievement of the first complete remission (CR). All parameters except cell proportion in G1/M cycle phase differed significantly with respect to histologic cell type, but were not related to the achievement of first CR. Inasmuch as the parameters significantly correlated with each other, multivariate discriminant analysis and proportional hazard regression were applied to estimate their discriminant/predictive values with respect to tumor malignancy. AgNORs proved to be far superior in all three clinical parameters, S-phase was significantly predictive for the achievement of first CR, and LS for tumor histology type. The statistical model applied narrowed down the analysis of seven parameters to two with respect to tumor histology type (AgNORs and LS) and achievement of first CR (AgNORs and S), but only to one for overall patient survival (AgNORs). Only the model for tumor histology type discrimination was statistically significant (R2 = 0.904, p < 0.001). It appears that AgNORs may be of utmost predictive importance for the clinical outcome in NHL.

Analysis of data on leukocyte surface markers for recognition of leukemia/lymphoma phenotype pattern.

OBJECTIVE: This study was designed to test the algorithm for the recognition of leukemia/lymphoma pattern, based on cell immunophenotype assessed using specific monoclonal antibodies and measured using flow cytometry. DESIGN AND METHOD: Analysis was performed by comparing phenotyping data with reference data, followed by scoring of such comparisons. Output of the recognition was designed as a report list of possible diagnoses (defined as objects in the informatic system). Reference data were compiled from the respective literature. RESULTS: From 57 blood and bone marrow samples tested in this study, accurate recognition of the real diagnosis (object) appeared on the first four places of the report list in 54 (94.7%) samples. CONCLUSION: The list of the objects recognized by the use of algorithm appeared to be helpful in making a differential diagnosis, occasionally pointing to the states that the physician had not in mind at the start of the analysis.

Natural killer cell number and activity in multiple sclerosis.

Natural killer (NK) cell percentage among cells appearing in lymphocytic flow-cytometric gate, and their concentration per unit volume of peripheral blood, as well as NK-cell in vitro activity, were determined in 45 patients with multiple sclerosis (MS, 34 with definite and 11 with probable diagnosis). Two age- and sex-matched control groups consisted of 27 healthy individuals and 19 individuals with other neurological diseases, respectively. NK cells were identified on the basis of their reaction with monoclonal antibody NKH-1, and NK-activity on the basis of their spontaneous killing of K-562 erythroleukemia target cells with mononuclear cells from peripheral blood. MS patients were analyzed in regard to the phase (active, stable inactive, stable progressive) and course (remittent, remittent-progressive, progressive) of the disease. In general, MS patients tended to have both lower number and activity of natural killer cells than either of the two control groups. A statistically significant decrease was found for the concentration of NKH-1+ cells in the blood of all MS patients, for the number of lymphocytes in the blood of patients with remittent course of the disease, and for the number of NKH-1+ cells in the blood of patients with progressive course of the disease. It appeared that more profound defects were associated with the progression of the disease; NK-cell number always appeared more affected than NK-cell activity. In MS patients, NK-cell activity correlated significantly with NK-cell percentages among lymphocytes but poorly with the concentration of NKH-1+ cells in the blood. In healthy controls, neither of the two correlations was significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Curricular and extracurricular activities of medical students during war, Zagreb University School of Medicine, 1991-1995.

War, as a major human disaster, affects many aspects of life, including medical education. This report describes curricular and extracurricular activities of the students at the Zagreb University School of Medicine during the wars in Croatia and neighboring Bosnia and Herzegovina. Although condensed versions of the curricula were prepared in case of a major breakdown in civilian life, the school maintained the continuity and quality of its curriculum throughout the war. Students engaged in extracurricular activities related to medical aspects of the war, including organization of resuscitation and first aid courses, collecting medical documentation on war victims, humanitarian help to refugees, and peace-promoting activities. Some students joined mobile surgical teams on the battlefronts. After army service, most of them returned to the school and successfully continued with their studies. The school also accepted guest-students from other new states emerged from former Yugoslavia. The authors found that the students' engagement in extracurricular activities related to medicine was enormously beneficial both to the psychological well-being of the students and to the region's peace-building efforts.

The effect of the zeolite clinoptilolite on serum chemistry and hematopoiesis in mice.

Zeolites are natural or synthetic crystalline alumosilicates with ion exchanging properties. Supplied in fodder, they promote biomass production and animal health. Our aim was to assess the effects of the natural zeolite, clinoptilolite, on hematopoiesis, serum electrolytes and essential biochemical indicators of kidney and liver function in mice. Two preparations differing in particle size were tested: a powderized form obtained by countercurrent mechanical treatment of the clinoptilolite (MTCp) and normally ground clinoptilolite (NGCp). Young adult mice were supplied with food containing 12.5, 25 or 50% clinoptilolite powder. Control animals received the same food ration without the clinoptilolite. After 10, 20, 30 and 40 days, six animals from each group were exsanguinated to obtain blood for hematological and serum for biochemical measurements as well as to collect femoral bone marrow for determination of hematopoietic activity. Clinoptilolite ingestion was well tolerated, as judged by comparable body masses of treated and control animals. A 20% increase of the potassium level was detected in mice receiving the zeolite-rich diet, without other changes in serum chemistry. Erythrocyte, hemoglobin and platelet levels in peripheral blood were not materially affected. NGCp caused leukocytosis, with concomitant decline of the GM-CFU content in the bone marrow, which was attributed to intestinal irritation by rough zeolite particles. The mechanically treated clinoptilolite preparation caused similar, albeit less pronounced, changes. In a limited experiment, mice having transplanted mammary carcinoma in the terminal stage showed increased potassium and decreased sodium and chloride levels, severe anemia and leukocytosis, decreased bone marrow cellularity and diminished content of hematopoietic progenitor cells in the marrow. The clinoptilolite preparations ameliorated the sodium and chloride decline, whereas the effects on hematopoiesis were erratic.

Hypersensitivity to pollen allergens on the Adriatic coast.

In central south Croatia, i.e., the Adriatic coast with the city of Split at the center, year-long pollen concentrations in the air were determined for typical local plants, and the area's plant pollination calendar was established. High concentrations of Parietaria officinalis pollen dominated during the year (up to 20% from April to June). Pollens of Pistacia lentiscus, Olea europaea, Pinus halepensis, Juniperus oxycedrus, Mimosa and Cistus monspeliensis were found in lower concentrations and for shorter time periods. Using both commercially available standard inhalation allergens and specifically prepared pollen allergens, skin testing was performed and the cause of hypersensitivity was determined in a population of 4116 atopic patients with respiratory symptoms. Some 38.8% of patients were allergic to standard pollen allergens (mixed grass pollen, mixed tree pollen, P. officinalis and Pittosporum tobira). Hypersensitivity to more than one allergen was found in 53% of patients, whereas 19% of patients did not react to any of the standard allergens. Additional testing with newly prepared individual allergens (P. lentiscus, O. europaea, P. halepensis, J. oxycedrus, Mimosa and C. monspeliensis) revealed the causes of hypersensitivity in a number of patients, but the testing of patients nonreactive to standard allergens still left 44% of these individuals without reaction to any of the allergens used. Hypersensitivity to the pollens of P. tobira, C. monspeliensis and J. oxycedrus is described for the first time.

Specific hyposensitization in patients allergic to Parietaria officinalis pollen allergen.

The effects of specific hyposensitization in 40 patients with Parietaria officinalis-sensitive seasonal rhinoconjunctivitis were studied during three years of treatment. The patients were treated with subcutaneous injections of a new, partially purified, characterized and standardized pollen extract of P. officinalis allergen (alum-absorbed depot preparation). Treatment was applied from November to mid March and it was clinically assessed during the plant flowering season (mid March to end of June). Laboratory tests were performed yearly when beginning and ending treatment. Serum concentrations of P. officinalis pollen allergen-specific IgE antibodies decreased (first year: from 18.7 +/- 7.7 to 17.9 +/- 7.6 PRU/ml; second year: from 16.3 +/- 7.1 to 14.1 +/- 6.6 PRU/ml; third year: from 12.3 +/- 5.6 to 10.9 +/- 5.6 PRU/ml) and those of specific IgG increased (first year: from 15.3 +/- 13.2 to 21.7 +/- 14.0%; second year: from 28.5 +/- 13.0 to 36.3 +/- 15.9%; third year: from 29.9 +/- 14.1 to 38.9 +/- 16.8%) during the treatment. Histamine release from peripheral blood leukocytes challenged in vitro with the allergen decreased during the three years of the treatment (first year: from 42.3 +/- 13.0 to 33.1 +/- 10.8%; second year: from 31.9 +/- 11.9 to 19.1 +/- 8.5%; third year: from 19.4 +/- 4.6 to 14.3 +/- 4.6%), whereas the size of skin test reaction and the percentage of eosinophils among white blood cells remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of three biological response modifiers on chemical carcinogenesis in mice.

The modulation of chemical carcinogenesis by three biological response modifiers was assessed in a mouse model. CBA mice given 20-methylcholanthrene s.c. were treated with peptidoglycan monomer, azure B and indomethacin for one month, either from day 0 or 75 after methylcholanthrene injection to assess their effects on tumor incidence (on days 150 and 300), time of tumor appearance, time of death, and duration and dynamics of tumor growth. All three agents significantly influenced some of the parameters of tumor growth, except tumor incidence on day 300. Highly significant sex differences in tumor appearance and growth were observed. Tumors with late appearance grew faster in comparison to tumors with early appearance. The data presented indicate that the effectiveness of anti-cancer body defense mechanisms can be best defined by the time of tumor appearance.

Gonadectomy abrogates sex differences in the effectiveness of chemical carcinogenesis in mice.

Sham-gonadectomized and gonadectomized male and female mice were given methylcholanthrene s.c. to assess the influence of sex hormones on carcinogenesis. Gonadectomy decreased the concentration of the respective sex hormone and increased the concentration of the opposite sex hormone. The results showed that androgens enhanced the effectiveness of carcinogenesis, while estrogens had the opposite effect. Gonadectomy effectively abrogated the sex differences in tumor induction.