A TL-based anthropomorphic benchmark for verifying 3-D dose distributions from external electron beams calculated by radiotherapy treatment planning systems.

Initial results are reported of a Polish-Finnish project to verify electron dose distributions calculated by treatment planning systems (TPSs), CadPlan v.6.3.2 and Theraplan v.3.5, which use different electron beam dose distribution algorithms. Treatment of gross tumour volumes representing lung and parotid cancer was simulated in an Alderson anthropomorphic phantom with thermoluminescent detectors (TLDs) (Li(2)B(4)O(7):Mn,Si) placed at selected measurement points inside its volume. The observed discrepancy between relative values of dose calculated and measured by TLDs at each of the measurement points and those calculated by the different TPSs at the same points is discussed.

Avidity and subclasses of IgG after immunization of infants with an 11-valent pneumococcal conjugate vaccine with or without aluminum adjuvant.

Finnish and Israeli infants received an 11-valent mixed-carrier pneumococcal conjugate vaccine with or without aluminum adjuvant at 2, 4, 6, and 12 months of age. The relative avidity of serotype 1-, 5-, 6B-, 14-, 19F-, and 23F-specific IgG antibodies in serum obtained at 7, 12, and 13 months of age was measured by EIA, using thiocyanate as a chaotropic agent. For all serotypes, except 14, avidity increased between the ages of 7 and 12 months. After boosting at 12 months, avidity further increased for all serotypes. The adjuvant improved antibody avidity against serotype 5. The IgG antibodies produced were mainly IgG1 subclass, although some infants also produced IgG2 after boosting. In conclusion, the immunization of infants with this 11-valent pneumococcal conjugate vaccine increased avidity of IgG, suggesting successful immunologic priming.

Activation of cell-mediated immunity following immunization with pneumococcal conjugate or polysaccharide vaccine.

The immunogenicity of pneumococcal polysaccharide (PS) vaccines can be improved by conjugating PS to a polypeptide carrier that alters the immune response from T-cell independent to T-cell dependent. In order to study the influence of PS or protein antigens as inducers of cell-mediated responses, 30 adults were immunized with a 23-valent pneumococcal PS vaccine (PS-group) or an 11-valent, tetanus and diphtheria mixed carrier conjugate vaccine with (adjuvant group) or without aluminium adjuvant (nonadjuvant group). Cell-mediated responses were analyzed on days 0, 14 and 28 after vaccination by measuring lymphocyte proliferation and production of interferon (IFN)-gamma (Th1 marker) or interleukin (IL)-4 and IL-5 (Th2 markers) cytokines after in vitro stimulation with the PS and protein components of the vaccines. Tetanus and diphtheria proteins were the main inducers of lymphocyte proliferative and cytokine responses. Conjugate vaccines induced increased proliferative responses to the tetanus or diphtheria protein, but not to the PS components. In the PS-group, a lymphocyte proliferative response to protein antigens was not observed. The number of antigen-specific and nonspecific IFN-gamma-secreting cells detected by ELISPOT tended to increase in all three groups in response to protein or to PS antigen. No major differences were detected in the number of IL-4-secreting cells measured 14 and 28 days after vaccination. The conjugate vaccine with adjuvant was associated with Th2 type of activation indicated by an enhanced IL-5 secretion in response to the tetanus and diphtheria protein antigens.

Tetravalent meningococcal polysaccharide vaccine is immunogenic in adult allogeneic BMT recipients.

Forty-four adult BMT recipients transplanted from an HLA-identical sibling donor were randomized to receive meningococcal polysaccharide (Men PS) vaccine either 8 (early group; 22 patients) or 20 (late group; 22 patients) months after BMT. The geometric mean concentrations (GMC) of antibodies to serogroup A Neisseria meningitidis (Men A) and serogroup C Neisseria meningitidis (Men C), determined by an EIA method, decreased during the first 6 months after BMT but remained at a stable level thereafter. Before vaccination the GMCs of anti-Men A were 1.53 microg/ml and 1.61 microg/ml, but 1 month after vaccination they were significantly higher, 3.46 microg/ml and 6.39 microg/ml, in the early and late groups. The GMCs of anti-Men C increased from 0.37 microg/ml and 0.44 microg/ml before vaccination to 3.31 microg/ml and 4.62 microg/ml at 1 month after vaccination in the early and late groups, respectively. By 6 months after vaccination the GMCs of Men antibodies had decreased to levels of about 50% of those measured at 1 month after vaccination. Two-fold responses to Men A PS were seen in 52% and 74% and to Men C PS in 76% and 89% of the BMT recipients in the early and late groups, respectively. Chronic GVHD had no influence on the vaccination response. In the present study, Men PS vaccine induced good and equal antibody responses to Men A and Men C PSs in allogeneic BMT recipients regardless of timing after BMT. Vaccination against Neisseria meningitidis should be considered, especially in the event of travelling or military service > or = 8 months after BMT.

Immunogenicity of pneumococcal conjugate vaccines.

BACKGROUND: Prevention of pneumococcal infections is a public health priority because of the high impact of the disease and because of the increasing problems due to antimicrobial resistance. Traditional vaccines, consisting of purified capsular polysaccharides (PSs) of Streptococcus pneumoniae, are not immunogenic in young children. In addition they confer only limited protection in patients with immunodeficiencies and hematologic malignancies. IMMUNOGENICITY OF PNEUMOCOCCAL CONJUGATE VACCINES: Immunogenicity of the PS vaccine has been enhanced by coupling pneumococcal PSs to proteins to produce a conjugate vaccine. Conjugate molecules are designed to possess T cell dependent properties, such as immunogenicity in early infancy, stimulation of high levels of IgG isotype antibodies and enhanced immunologic memory responses. In the clinical studies multivalent pneumococcal conjugate vaccines have been shown to induce an IgG-dominating serum antibody response against common pneumococcal serotypes causing infections in children. A booster dose later in life creates a robust and rapid antibody response, indicating the existence of immunologic memory in primed children. Antibodies induced by conjugate vaccines are functionally active, as demonstrated by their high avidity and opsonophagocytic activity.

RT-PCR from eosinophil-depleted leukocytes without RNA extraction: cell selection using streptavidin PCR tubes.

OBJECTIVES: The major RNase activity of leukocytes has been attributed to eosinophil-derived neurotoxin EDN. Depletion of eosinophils enables RT-PCR from 10(5) leukocytes without RNA extraction. In this study we introduced streptavidin-coated PCR tube strips for the selection of eosinophil-free leukocytes for RT-PCR analysis. DESIGN AND METHODS: Polypropylene 0.2 ml PCR tube strips were coated with streptavidin and biotinylated antibodies against cell surface antigens were attached to the tubes. CD7-positive T-lymphocytes, CD19-positive B-lymphocytes and CD16-positive cells (mainly neutrophils and monocytes) were positively selected by incubating of 1-2 x 10(5) leukocytes in the antibody-coated PCR tubes for 30 min at 23 degrees C. RESULTS: The mean amount of cells bound into a tube was 31,500 (CV25%) T-cells and 8,600 (CV61%) B-cells from 12 blood samples, and 23,600 (CV22%) CD16+ cells from 17 samples. The influence of selected cell lysate on the RT-PCR analysis of Philadelphia chromosome (bcr/abl translocation) from 100 K562 cells was small: 78% (CV28%) of the leukocyte-free signal was obtained in the presence of CD16+ cells or 89% (CV15%) and 99% (CV11%) and in the presence of T-cells and B-cells, respectively. CONCLUSIONS: These results suggest that through the introduction of eosinophil-free cell population into RT-PCR a reproducible method with reasonable leukocyte yield and avoiding RNA extraction was developed.

Major interference from leukocytes in reverse transcription-PCR identified as neurotoxin ribonuclease from eosinophils: detection of residual chronic myelogenous leukemia from cell lysates by use of an eosinophil-depleted cell preparation.

BACKGROUND: The extraction of RNA from leukocytes for reverse transcription-PCR (RT-PCR) is time-consuming and contributes to variation in analysis of the Philadelphia (Ph1) chromosome of chronic myelogenous leukemia (CML) by RT-PCR. To detect residual CML after bone marrow transplantation, mRNA from at least 10(5) leukocytes should be analyzed, but the RNase activity of the cells precludes simple leukocytes lysis as an alternative to RNA extraction. We sought to identify the main source of RNase activity of leukocytes. METHODS: We used a three-step chromatographic process and amino acid sequence analysis. We selected eosinophil-free granulocytes by using a biotinylated CD16 antibody and selected mononuclear cells by fractionating the leukocytes with a Ficoll-Paque(R) density gradient. RESULTS: Chromatography and amino acid sequencing identified eosinophil-derived neurotoxin (EDN) as the main source of leukocyte RNase. Depletion of eosinophils reduced the EDN content of cell lysates by approximately 90%, allowing a signal from a lysate of 50 K562 Ph1-positive cells mixed with 10(5) CD16(+) granulocytes that was equivalent to 77% of the signal in the absence of leukocytes. A similar lysate with mononuclear cells gave a signal equivalent to 53% of that without mononuclear cells. RNA extraction gave a signal equivalent to only 24% of the leukocyte-free control. CONCLUSION: The depletion of eosinophils during the preparation of leukocyte samples for RT-PCR efficiently reduces the risk of mRNA degradation by ribonucleases, enabling RT-PCR analysis directly from cell lysates with a better signal than can be obtained by RNA extraction.

Immune responses and clinical outcome of massive human osteoarticular allografts.

Cell mediated immune responses as measured by lymphocyte proliferation induced by the mitogens phytohemagglutinin and concanavalin A and antigen extracts of donor derived bone were studied within 2 years after wide resection of bone tumors in 18 patients receiving fresh frozen massive osteoarticular allografts. No uniform changes were seen in mitogen induced responses in 18 patients. However, five of nine patients tested with antigen extracts of donor derived bone showed elevated immune responses, one moderate and four weak. The incorporation of the allograft (evaluated by repeated radiographs; specific isotope techniques; clinical outcome assessed by the functional rating scores of Mankin-Waber and the Musculoskeletal Tumor Society; and histologic biopsy findings on new bone formation) did not differ in these patients from those in patients without any response to donor derived tissue. During a long term followup (mean, 11 years; range, 2-20 years), degenerative joint and sclerotic density bone changes developed after 2 to 4 years without correlation to immune responses. Histologic specimens showed no signs of immunologic reaction, and no clinical rejection episodes were recorded. A slightly variable immune response to allograft bone seems to occur, but its clinical significance for outcome of the grafts remains to be determined. The low immune responses might reflect a low antigen release rate through an indirect pathway or immunologic tolerance to antigens or proteins shed from massive allografts that are nonliving scaffold implants during the creeping substitution process, corresponding to the low immune response and the slow histologic repair.

A randomized comparison between early and late vaccination with tetanus toxoid vaccine after allogeneic BMT.

Forty-five adult HLA-matched sibling BMT recipients were randomized to receive tetanus toxoid (TT) vaccine at 6, 8 and 14 months (early group, n = 23) or at 18, 20 and 26 months after BMT (late group, n = 22). At 6 months after BMT, prior to the first vaccination, 90% of the early group patients had a protective tetanus antibody concentration of > or = 0.1 HU/ml (passive haemagglutination test) but only 70% of the late group patients did so at 18 months after BMT. The antibody responses 1 month after each of the three TT vaccine doses were similar in the two vaccination groups, except that after the first dose, four-fold responses occurred more frequently in the late group. All vaccinated patients achieved the protective antibody concentration of > or = 0.1 HU/ml. In the late group the recipient antibody responses after the first and second vaccine doses correlated with the donor anti-TT level. A tetanus vaccination schedule consisting of three vaccine doses is equally immunogenic when started at 6 or 18 months after BMT. Donor immunity against tetanus may influence recipient responses to TT vaccination.

Prognosis of acute otitis media: factors associated with poor outcome.

Factors associated with poor outcome of acute otitis media (AOM) were analysed in 131 children aged 1/4 to 7 1/2 (median 2 1/2) years. After AOM, altogether 37 (28%) of the children had poor outcome: 15 children (12%) clinical failure (unimprovement or worsening of pre-treatment signs and symptoms within 2 weeks of onset of therapy) and 31 (24%) persistent middle ear effusion (MEE) > or = 1 month post-treatment. Of the different variables studied in multivariate analysis, age < 2 years (p < 0.01), history of allergic skin or respiratory symptoms (p = 0.02), > or = 6 h duration of pre-treatment earache (p = 0.01) and B. catarrhalis in MEE (p = 0.05) were associated with clinical failure. Children with previous adenotomy or unilateral AOM had no failures. Persistence of MEE at 1 month was associated with age < 2 years (p = 0.05), otitis proneness (p = 0.03), bilaterality of AOM (p < 0.01) and S. pneumoniae in MEE (p = 0.01) in univariate but not in multivariate analysis.

A comparison of early and late vaccination with Haemophilus influenzae type b conjugate and pneumococcal polysaccharide vaccines after allogeneic BMT.

Forty-five adult allogeneic BMT recipients were randomized to receive Haemophilus influenzae type b (Hib)-diphtheria toxoid conjugate vaccine (PRP-D) at 6 and pneumococcal polysaccharide vaccine (Pnc PS) at 8 months (early group; n = 23) or at 18 and 20 months (late group; n = 22) after BMT, respectively. Serum antibody concentrations against Hib and Pnc PS types with varying immunogenicity (serotypes 3, 6B, and 19F) were measured by ELISA. The responses at 1 month after vaccination with PRP-D and Pnc PS were poor and similar in the two vaccination groups, except that two-fold responses in the concentration of antibodies to the most immunogenic Pnc serotype 3 occurred more frequently in the late group. In the late group recipients, the antibody responses elicited by Pnc serotypes 3 and 19F correlated with the concentrations of the corresponding antibodies in the donor. This study on allogeneic BMT recipients shows that vaccination at 6-8 months after BMT with one dose of PRP-D and Pnc PS vaccine is as immunogenic as vaccination at 18-20 months after BMT, and suggests that donor immunity to Pnc PS affects recipient responses to Pnc PS vaccination. Consequently, as among allogeneic BMT recipients the greatest risk for infections by encapsulated bacteria occurs during the first 1-2 years after BMT, Hib and Pnc vaccines should not be given later than 6-8 months post-BMT.

Epidemiological views into possible components of paediatric combined vaccines in 2015.

Efforts to develop combined vaccines for childhood immunization schedules will during the next years focus on combined administration of the existing vaccines which have already shown their impact. First candidate components for more wide-spectrum combinations could be the new antigens against severe invasive infections caused by encapsulated bacteria. Multivalent pneumococcal and meningococcal group A and C conjugate vaccines are already in clinical trials, and the same is true of the first candidates for the meningococcal group B vaccine. Pneumococcal conjugate vaccines are also important in prevention of a large variety of respiratory infections. Since viruses are important causative agents of bronchiolitis and pneumonia, components of the paediatric combined vaccine should include at least respiratory syncytial virus, and possibly also other respiratory viruses like parainfluenza and adenoviruses. The third group of diseases to be considered from the preventive point of view are congenital infections, and vaccines against herpes simplex viruses, cytomegalovirus, or group B streptococci might be included in a combined vaccine to be administered to adolescents in order to afford protection to their future children.

Detection of Philadelphia chromosome using PCR and europium-labeled DNA probes.

More than 95% of the patients with chronic myelogenous leukemia (CML) carry translocations between protooncogene abl of chromosome 9 and bcr gene of chromosome 22, resulting in the Philadelphia chromosome (Ph1). After allogeneic bone marrow transplantation (BMT) it is important to detect possible residual malignant cells in CML patients. A new sensitive hybridization method combined with polymerase chain reaction (PCR), based on the detection of the europium (Eu3+) label by time-resolved fluorescence, was applied for the detection of Ph1 chromosome. Total RNA from 10(6) peripheral blood leukocytes was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction. After cDNA synthesis by reverse transcriptase, the PCR amplification (30 cycles) was carried out. In the detection phase two oligonucleotide probes were used in the hybridization reaction, one biotinylated (bcr gene, exon 2) and one (abl gene) labeled with Eu3+. The hybrids were collected in a streptavidin-coated microtitration well and the bound Eu3+ was measured in a time-resolved fluorometer. To assess the sensitivity of the method, different numbers of CML cell line K562 cells were mixed with 10(5) apparently normal human leukocytes. Five K562 cells/10(5) leukocytes could be detected. Six patients with CML confirmed by clinical and cytogenetic criteria were studied. Three of the patients underwent an allogeneic BTM 6-18 months before the investigation and all of them were Ph1-negative. The other three patients who were nontransplanted were positive as expected.

Preceding respiratory infection predisposing for primary and secondary invasive Haemophilus influenzae type b disease.

Cases (117) with invasive Haemophilus influenzae type b (Hib) disease and their family members reported symptoms of respiratory infection during the 4-week period before the onset of Hib disease significantly more often than age-, sex- and residence-matched controls (225) and their family members during the same time period. Viral (adenovirus; influenza A and B; parainfluenza types 1, 2 and 3; and respiratory syncytial virus) and Mycoplasma pneumoniae serology was performed in 84 paired sera from cases and 112 paired sera from controls, who were healthy children matched to the cases by age, year and season. Viral or M. pneumoniae infection was diagnosed equally often among cases and controls (18% for both groups). However, patients who were associated cases of Hib disease (i.e. either the primary or secondary case of a case pair) had a diagnostic viral serology more often (50%) than did sporadic cases (13%) (odds ratio, 7.0; 95% confidence interval, 1.6 to 33; P = 0.006). These results suggest that some infectious agent(s) caused symptoms among the patients and circulated among the patients' closest contacts immediately before their development of Hib disease and possibly predisposed for invasive Hib disease. For the development of associated Hib disease among close contacts of an index case, adenovirus, influenza A, respiratory syncytial virus or para-influenza type 1, 2 and 3 infections may be important.

Clinical scoring system in the evaluation of adult pharyngitis.

OBJECTIVE: To compare results of a clinical scoring system for diagnosis of group A streptococcal pharyngitis with microbiologic results, when several different pharyngeal pathogens were tested simultaneously. DESIGN: Evaluation of clinical manifestations of 106 adult patients with pharyngitis of different microbial origin. SETTING: General private practice; Health Center Pulssi, Turku, Finland. PATIENTS: Adult patients whose chief complaints were sore throats. MAIN OUTCOME MEASURE: A symptom score that was assigned to each patient according to the total number of certain signs and symptoms that are postulated to increase the probability of group A streptococcal pharyngitis and blood measurements for infection. RESULTS: The highest symptom scores, 3 and 4, were found in 21 patients. These patients had pharyngitis due to group A streptococcus (four patients), group C streptococcus (four patients), group G streptococcus (two patients), group F streptococcus, Mycoplasma pneumoniae, Chlamydia pneumoniae, influenza A virus, influenza B virus, herpes simplex type 1 virus (two patients), and coxsackie B4 virus. No pathogen could be identified from three of the 21 patients. The C-reactive protein values and the leukocyte counts were raised significantly more often in streptococcal infections than in infections of other origin; the P values were .00016 and .028, respectively. CONCLUSION: Use of a clinical scoring system alone for diagnosis of pharyngitis may lead to improper use of anti-microbial agents. There is a need for accurate microbiologic diagnostic procedures in general practice to determine proper treatment of pharyngitis as well as to test the effect of antibacterial and, in the future, antiviral treatment in respiratory tract infections.

Application of a new monoclonal antibody for time-resolved fluoroimmunoassay of human pancreatic phospholipase A2.

A monoclonal antibody, designated 2E1, against human pancreatic phospholipase A2 was produced by hybridization of myeloma cells with spleen cells of immunized BALB/c mice. The hybridomas were screened for antibody production by time-resolved fluoroimmunoassay (TR-FIA). The antibody was found to belong to subclass I of murine IgG. The specificity of the antibody was confirmed by immunohistochemistry of pancreatic and other tissues, by immunoblotting of a crude aqueous extract of human pancreas and purified human pancreatic phospholipase A2 and by TR-FIA. A solid-phase time-resolved fluoroimmunoassay was developed by using the monoclonal anti-phospholipase A2 antibody as the catching antibody and a polyclonal sheep anti-phospholipase A2 antibody labelled with europium as the detecting antibody. The validity of the new TR-FIA of human pancreatic phospholipase A2 was confirmed by using it to measure the phospholipase A2 concentrations in serum samples from healthy subjects and from patients suffering from acute pancreatitis.

The causes of hospital-treated acute lower respiratory tract infection in children.

OBJECTIVE: To determine the etiologic agents in children with acute lower respiratory infection. DESIGN: A survey of a series of patients. SETTING: General pediatric hospital serving an urban population with and without referrals in Helsinki, Finland. PARTICIPANTS: 135 Finnish children aged 2 months to 15 years (mean, 1.75 years), with clinically defined acute lower respiratory infection (with difficulty of breathing), or found to have fever and a pneumonic infiltrate on chest roentgenogram. SELECTION PROCEDURES: Consecutive sample on voluntary basis. INTERVENTIONS: None. MAIN RESULTS: Of 121 children with adequate samples, an etiologic diagnosis could be established in 84 (70%): 30 (25%) had bacterial, 30 (25%) viral, and 24 (20%) mixed infections. Antibody assays alone identified the agent in 91% of positive cases. CONCLUSIONS: Bacterial infections are common but generally underestimated in acute lower respiratory infection; serologic methods add significantly to their detection.

Serum tumor necrosis factor-alpha concentrations in children hospitalized for acute lower respiratory tract infection.

Tumor necrosis factor-alpha (TNF alpha) concentrations were measured by radioimmunoassay in sera of 118 children (median age, 1.7 years; range, 2 months-15 years) hospitalized for acute lower respiratory tract infection (ALRI). Both viral and bacterial ALRI were associated with elevated concentrations of TNF alpha. Concentrations greater than 40 ng/l were seen in children with bacterial or mixed ALRI in 64% and with viral ALRI in 50% of cases. Elevated concentrations were associated with longer duration of fever before admission (P less than .05) and with a higher serum C-reactive protein concentration (P less than .05). There were no significant differences in TNF alpha concentrations between gram-positive and gram-negative infections, nor was there an association with clinical severity of ALRI. TNF alpha concentrations decreased in most patients to normal within 5 days of hospitalization, irrespective of the etiology of the infection.

High volume lesser sac lavage in acute necrotizing pancreatitis.

The effect of lesser sac drainage with or without lavage on some early predictors and on outcome in acute necrotizing pancreatitis was analysed. The evaluation was made prospectively for 24 patients, in a single centre study. According to Ranson's criteria and laparotomy findings, the lavage and drainage groups were comparable and the pancreatitis was severe and necrotizing in both groups. In a longitudinal analysis of the first 4 postoperative days, lavage did not show any advantage over drainage, as measured by seven prognostic signs (serum creatinine, blood glucose, base excess, haematocrit, white blood cells, C-reactive protein and immunoreactive phospholipase A2 concentration). Furthermore, the study did not find that lavage had any positive effect on the incidence of mortality (36 versus 17 per cent in the drainage group) or on septic complications in acute necrotizing pancreatitis. In the total series the extent of pancreatic necrosis was an essential predictor of the outcome.