Clara cell secretory protein: determination of serum levels by an enzyme immunoassay and its importance as an indicator of bronchial asthma in children.

Clara cell secretory protein (CC16) is a 16kDa protein secreted by Clara cells in the lining fluid of bronchiolar and bronchial epithelium. CC16 presents several biologic properties, and has been shown to have immunomodulatory and anti-inflammatory activity. It may play a role in controlling inflammation in the airway. There is some evidence that the CC16 level is primarily lower in adult individuals with bronchial asthma, thus contributing to its pathophysiology. This study was designed to examine CC16 serum levels of children, healthy and with asthma. An enzyme solid phase immunoassay utilizing monoclonal antibody to CC16 was the analytical method to determine the protein concentration in blood sera. The method showed excellent linearity, high sensitivity (detection limit: <50 ng/l) and precision. It was found that asthmatic children appear significantly lower levels (P < 0.001) of CC16 in serum as compared to healthy ones. It is, therefore, concluded that CC16 may be a useful diagnostic index of bronchial asthma in the early child-age.

Study of minimal residual disease in B-cell lineage acute lymphoblastic leukemia of childhood, using clone-specific probes.

In B-cell lineage acute lymphoblastic leukemia (B-ALL), the clonal rearrangements of the immunoglobulin heavy chain gene locus (IgH), can be used as a molecular marker for the detection of minimal residual disease (MRD). Patients in complete remission may still harbor leukemic cells undetectable by conventional methods such as light-microscopic examination, immunophenotyping and cytogenetics. 30 children with B-ALL were screened at diagnosis by polymerase chain reaction (PCR) for their IgH gene repertoire. 7/30 patients were extensively studied using patient-specific oligonucleotide probes derived from the sequence analysis of bone marrow (BM) samples at diagnosis. 210 PCR products from follow-up BM samples corresponding to these 7 patients were hybridized with the appropriate clone-specific probe in order to detect MRD with high sensitivity and specificity. All the patients were in morphological remission during and after therapy. 25/30 patients were PCR positive at diagnosis. 4/7 patients who were examined for MRD had detectable disease in various periods after diagnosis. Molecular signs of residual cells can persist for a long time during and after therapy. Long term follow-up of MRD could determine the period of therapy and predict relapse, indicating therapeutic interventions.

Presence of the bcr/abl rearrangement in a patient with chronic neutrophilic leukaemia.

An 83 year old women presented with a myeloproliferative disorder involving the myeloid and megakaryocytic lines, and characterised by mature neutrophil leucocytosis. There was a high/normal neutrophil alkaline phosphatase activity and absence of the Philadelphia chromosome, features compatible with a diagnosis of chronic neutrophilic leukaemia (CNL). Southern blot analysis of the patient's DNA revealed the presence of the bcr/abl rearrangement. Combined with a previous report of detection of Ph1 chromosome in long term bone marrow cultures in a patient with CNL, this finding suggests that the bcr/abl hybrid gene might occasionally result in a myeloproliferative disorder with a phenotype closely resembling that of CNL.

Activity of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase in primary brain tumors in children.

The activity of the enzymes 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase has been studied cytochemically in children's primary brain tumors. These enzymes play a significant role in purine biosynthesis. Thirty children, aged 1-12 years, were studied, 12 with medulloblastoma, 14 with glioma grade I-IV, and 4 with ependymoma. The activity of the enzymes was apparent as cytoplasmic granules that sometimes overlie the nucleus of the tumor cells. This coincidence showed that different types of brain tumors exhibit different degrees of enzymic activity, which in some cases correlated positively with the malignant potential of the tumor. Approximately one third of the cases were negative for any activity of these enzymes. The intensity of the staining of 5,10-methenyl tetrahydrofolate cyclohydrolase activity was actually higher than that of 5-formyl tetrahydrofolate cyctodehydrase. The clinical or prognostic significance of these findings remains to be clarified, but we believe that cylochemistry provides a sensitive technique for the detection, localization, and description of these enzymes in brain tumor cells. A clear understanding of the mode of action of these enzymes may contribute to devising novel therapeutic strategies.

Study of apoptosis in peripheral blood of patients with acute lymphoblastic leukemia during induction therapy.

In acute lymphoblastic leukemia (ALL) the apoptosis of blast cells in peripheral blood (PB) and bone marrow before and/or during treatment, is of great interest. As the morphological changes during apoptosis provide the most reliable markers, in the present study we utilized a nuclear stain based on ethidium bromide (EtBr) for the rapid qualitative and quantitative measurement of circulating apoptotic cells directly in PB suspensions without fractionation. By using a fluorescent microscope the apoptotic cells appeared clearly visible, making the estimation of their percentage straightforward. We studied apoptosis before and during the onset of chemotherapy in PB from 16 children with ALL at diagnosis, and one upon relapse. In the cases studied at diagnosis the circulating apoptotic cells were found in variable percentages after 24 hours of treatment. Maximal apoptosis was observed after 24 hours of treatment in five cases and after 48 hours in two cases. After 96 hours of treatment the cases studied at diagnosis could be divided into three groups: those with a) negligible apoptotic cells, b) between 8% and 12% apoptotic cells and c) a high percentage of apoptotic cells (more than 20%). The relapsed case was characterized by P-glycoprotein positive blast cells, and circulating apoptotic cells which remained very low at all time points. Thus, it is possible to evaluate the response to treatment by studying apoptosis directly in peripheral blood. Therefore, the maximum apoptotic effect and the percentage of circulating apoptotic cells at the different time intervals must be considered.

Effect of trimethoprim-sulphamethoxazole in Langerhans' cell histiocytosis: preliminary observations.

Twenty-three children with Langerhans' cell histiocytosis (LCH) have been treated with trimethoprim-sulphamethoxazole (T-S) in a 4-year period. The children are classified in two main groups according to the extent of their disease. Group A includes 16 children with single system disease and group B, seven children with multisystem disease. All patients were treated for 4 weeks to 3 months. The results of treatment are evaluated in terms of response in individual organs involved. All children with single system disease had a good response to the drug. Children with multisystem disease had a good response to some organs but a poorer outcome for the lungs and for the blood. These patients did not respond even to conventional chemotherapy.

Analysis of BCL2 and MYC expression in non-Hodgkin's lymphomas by in situ hybridization: correlation with chromosome translocations.

We have used an in situ hybridization method for analysis of expression of BCL2 and MYC on cytospun preparations of normal and malignant lymphoid cell lines and tissue sections of normal and malignant lymph nodes. The probes comprised 50-mer antisense oligonucleotides starting at the ATG codons of exon 3 of BCL2 and exon 2 of MYC. We studied the expression of these two genes in frozen tissue sections of biopsy specimens derived from normal and hyperplastic lymph nodes, B-cell lymphomas carrying the t(14;18)(q32;q21) and t(8;14)(q24;q32) translocations, and T-cell lymphomas with clonal chromosome abnormalities. While all proliferating cells expressed both genes, BCL2 expression was increased two- to threefold in follicular lymphomas with t(14;18) and MYC expression was increased two- to four-fold in high-grade lymphomas with t(8;14). These results are consistent with previous data on deregulated expression of these genes obtained from study of lymphoma cell lines carrying the relevant translocations.

Distinct patterns of chromosome abnormalities characterize childhood non-Hodgkin's lymphoma.

We report here cytogenetic studies of a series of 23 childhood non-Hodgkin's lymphomas (NHL), a group that has previously not been subjected to detailed cytogenetic analysis. Combining our results with data from 25 tumours in the published literature, we have performed the first cytogenetic analysis of a large series of childhood NHL. Our results show that the cytogenetic changes encountered in NHL of children are distinct and may be different from those seen in NHL of adults reflecting the previously recognized differences in histological presentation and clinical behaviour of the two entities. Thus, the most frequently occurring translocation in B-cell lesions in children was t(8;14)(q24;1q32). Other translocations frequently seen in adults such as t(14;18)(q32;q21),t(11;14)(q13;q32) and t(3;22)(q27;q11) were either rare or so far not seen in children, although reciprocal translocations appeared to be generally prevalent in childhood NHL. Combining our data with those in the published literature, we have identified two new recurring translocations [t(1;17)(p36;q21) and t(1;14)(p36;q22)], and a recurring duplication [dup(11)(q13;q23)] in this group of lymphomas. In addition, our literature survey identified a third recurring translocation [t(5;14)(q23;q32)] which was previously reported in two cases of childhood NHL. Our analysis also showed differences in the types of nonrandom translocations between childhood NHL and acute lymphoblastic leukaemia (ALL) in children suggesting that biologically these entities are different from one another. This study thus uncovers patterns of chromosome change associated with childhood lymphoma thus providing new opportunities for investigation of their clinical significance by correlation analysis and biological significance by molecular analysis.

Structural alterations in the 5' region of the BCL2 gene in follicular lymphomas with BCL2-MBR or BCL2-MCR rearrangements.

We report a new class of molecular lesions in the 5' region of the BCL2 protooncogene when it undergoes a t(14;18) translocation-associated rearrangement in the major break cluster (MBR) or minor break cluster (MCR) regions. Among 52 tumors assayed for BCL2 rearrangements using the MBR, MCR, and 5' probes, seven (six with MBR and one with MCR translocation breaks) showed aberrant bands in unique enzyme digests of DNA hybridized with the 5' probe. The aberrant bands in four tumors were in HindIII digests, in two they were in EcoRI digests, while the aberrant band in one was in a BamHI digest. The two EcoRI bands and two of the four HindIII bands were of identical sizes. Germline polymorphisms as the source of these bands was ruled out by appropriate control experiments. These results suggest that the bands were derived from point mutations or small deletions in the 5' region of BCL2. The significance of this alteration to BCL2 function in translocation-carrying tumors remains to be determined.