Cells In Pediatric Patients Research Articles

Evaluation of Antibody Response to Polysaccharide Vaccine and Switched Memory B Cells in Pediatric Patients with Inflammatory Bowel Disease

Background/AimsInflammatory bowel disease (IBD) is a chronic disease of the gastrointestinal tract, whose etiologies are still unknown. This study was performed to evaluate the humoral immune response in terms of B cell functions in selected IBD patients.MethodsEighteen pediatric patients with IBD, including 12 cases of ulcerative colitis (UC) and six with Crohn disease (CD), were enrolled in this study. The pneumococcal vaccine was injected in all patients, and the IgG antibody level to the polysaccharide antigen was measured before and 4 weeks after injection. The B cell switch-recombination process was evaluated.ResultsFive patients with IBD (three CD and two UC) had defects in B cell switching, which was significantly higher than in controls (p=0.05). Ten patients had a specific antibody deficiency and exhibited a higher frequency of bacterial infection than the healthy group. The mean increased level of IgG after vaccination was lower in IBD patients (82.9±32.5 µg/mL vs 219.8±59.0 µg/mL; p=0.001). Among the patients who had an insufficient response, no significant difference in the number of switched memory B-cell was observed.ConclusionsA defect in B lymphocyte switching was observed in pediatric IBD patients, and especially in those patients with CD. Owing to an increased risk of bacterial infections in those patients with antibody production defects, pneumococcal vaccination could be recommended. However, not all patients can benefit from the vaccination, and several may require other prophylactic methods.

Authors' Response

To the Editor: We thank Dr Roggenbuck et al (1) for the interest in our recent article on pancreatic autoantibodies and autoantibodies against goblet cells in pediatric patients with inflammatory bowel disease (IBD). In our study, conducted only in pediatric patients with IBD, the combined prevalence of exocrine pancreatic antibody (PAB) and recombinant pancreatic antibody (rPAB) was significantly higher in pediatric patients with Crohn disease (CD) (34% and 35.9%) and in pediatric patients with ulcerative colitis (UC) (20.4% and 24.5%) compared with controls. In our study, the presence of PAB and rPAB was not associated with disease phenotype in either CD or UC (1). It should be noted that Bogdanos et al (2) enrolled a mixture of patients with IBD up to 87 years; only 18.3% of the CD patients were in A1 group (younger than 17 years) and indeed, there were no patients with UC younger than 17 years. Therefore, it is difficult to compare the data of the 2 studies; hence, age may affect location and behavior also. In our patients with CD, the presence of PAB and rPAB (34% and 35.9%) was comparable with the results of Bogdanos et al (30.2%). In UC, we found higher prevalence of rPAB and ASCA (24.5% and 26.5%) than Bogdanos et al (8.8% and 6.9%). These differences may be because of the younger age of our patients with IBD (median age 13.9 vs 36 years [CD] and 12.5 vs 47 years [UC]) and the higher percentage of sclerosing cholangitis in our patients with UC (20.9%) (3), although in other studies similar prevalence of PAB was noted in UC (22.7% (4) and 23.3% (5)). It should be noted that Bogdanos et al found relative low antibodies against Saccharomyces cerevisiae prevalence in their CD group (35.5%). Based on previous studies, there are conflicting results of association between PAB and CD phenotype (2,4,5). In agreement with the results published by Bogdanos et al (2), we observed numerically higher PAB and rPAB positivity in patients with ileocolonic CD (60% and 59.4%) than in patients with colonic (28.6% and 27%) and ileal disease (11.4% and 13.5%); however, the difference did not reach significance. Regarding CD behavior we could not demonstrate any relation, perhaps because of relatively higher proportion of patients with B1 phenotype and use only indirect immunofluorescence (IIF) method. We agree with Bogdanos et al that the combined use of IIF and enzyme-linked immunosorbent assay may improve the sensitivity of PAB. Further simultaneous multicentric studies conducted in pediatric and adult patients with IBD are necessary to investigate anti-glycoprotein 2 and anti-CUB and zona pellucida–like domain-containing protein 1 and their phenotype association separately using either IIF or enzyme-linked immunosorbent assay.

485 Regulatory T Cells Subsets in Children with Sle

In the case of SLE, Treg deficiencies have been described in mouse models of SLE. However, there are somehow conflicting data in the literature on whether Treg cells in human...

Quantitative Analysis of Naturally-Occurring Regulatory T Cells in Pediatric Patients with Systemic Lupus Erythematosus

Quantitative Analysis of Naturally-Occurring Regulatory T Cells in Pediatric Patients with Systemic Lupus Erythematosus

Defective calcium signaling and disrupted CD20–B-cell receptor dissociation in patients with common variable immunodeficiency disorders

B cells of patients with common variable immunodeficiency (CVID) disorders display impairment in production of immunoglobulin class-switched antibodies, which is possibly contributed to by defects in early B-cell activation. On resting B cells, B-cell receptors (BCRs) are organized in oligomers that are signaling inactive. Their triggering by cognate antigen causes the lateral reorganization of BCRs and associated proteins into signalosomes, resulting in BCR-activated calcium entry. In resting cells the B-cell surface antigen CD20 is associated with the BCR but dissociates on signalosome formation. We sought to determine whether CD20 dissociation from the BCR during early B-cell activation might contribute to the development of CVID disorders. We evaluated BCR signalosome formation, internalization, and signaling in primary B cells of pediatric patients with CVID disorders and healthy control subjects. In many pediatric patients with CVID disorders, Bcells exhibit significant deficits in BCR triggering-mediated calcium entry in the cytosol, which correlates with impaired plasmablast differentiation invitro. These alterations did not originate from upregulation of CD22 or defects in calcium channels and did not involve gene mutations in phospholipase Cγ2 or Bruton tyrosine kinase. Instead, B cells from patients with CVID disorders exhibited reduced BCR dissociation from CD20. BCR or CD20 cross-linking induced less BCR internalization, and antibody-mediated CD20 triggering elicited less BCR downstream signaling, as measured based on secondary fluxes. We propose that CD20 dissociation from the BCR signalosome is pivotal to BCR-mediated calcium mobilization in the cytosol. Defects in CD20/BCR signalosome conformation might predispose to the spectrum of CVID disorders.

Mobilization and Harvesting of Peripheral Blood Stem Cells in Pediatric Patients With Solid Tumors

Survival of patients with high-risk pediatric solid tumors has improved with the introduction of a high-dose chemotherapy regimen and autologous stem cell rescue. Here, we present our data regarding the evaluation of the efficacy and safety of hematopoietic stem cell mobilization and harvesting in children with solid tumors. From November 2002 to March 2010, 85 children underwent autologous peripheral blood stem cell collection; 35 (41.1%) of them weighed less than 20 kg and were diagnosed with neuroblastoma, Wilms' tumor, medulloblastoma, yolk sac sarcoma, or non-Hodgkin's lymphoma. The mobilization regimens included disease-specific chemotherapy plus granulocyte colony-stimulating factor in most of the patients. The median age and weight at the time of apheresis was 36 months and 13.5 kg, respectively. Large-volume leukapheresis was performed with the aim of reducing the psychological and financial impact of leukapheresis by reducing the number of procedures while collecting a large number of cells. The median number of mobilization and leukapheresis procedures per case was one. The pre-apheresis CD34+ cell count ranged from 2 to 845 µL, with a median of 24 µL. A median of four patient blood volumes was processed per procedure, lasting 279 min (range, 113-420 min). A radial catheter was used for harvesting in 35 procedures (71.4%). The median yield of CD34+ cells was 6.6×10(6) /kg per patient. The targeted dose of 5×10(6) /kg CD34+ cells was realized in 80% of patients. The tolerance of peripheral blood stem cell collection in our patients was good. In conclusion, the collection of peripheral blood stem cells is an effective and safe procedure, even when conducted on the youngest children.

Significance of detecting the EBV-DNA level in peripheral blood mononuclear cells and the EBV-infected cell type in patients with chronic active EBV infection

To study the difference in the EBV-DNA level in peripheral blood mononuclear cells (PBMC) and the type of Epstein-Barr virus (EBV)-infected cells in pediatric patients with chronic active EBV (CAEBV) infection, acute EBV infection (AEBV) and healthy children, and to analyze the relationship between the above difference and the clinical manifestation of CAEBV. Real-time fluorescent quantitative polymerase chain reaction (PCR) was used to detect the EBV-DNA levels in peripheral blood mononuclear cells (PBMC) in 12 normal children, 10 pediatric patients with CAEBV infection and 13 pediatric patients with AEBV infection in our hospital between March 2004 and April 2008. Immunomagnetic bead cell fractionation and fluorescent in situ hybridization (FISH) by EBV encoding RNA-1 ( EBER-1) probe were used in the healthy children, EBV-DNA positive CAEBV patients and AEBV patients to detect the type of EBV-infected cells. The average EBV-DNA level in CAEBV patients' PBMC was (6.8 x 10(7) +/- 1.1 x 10(8)) copies/ml, while the average EBV-DNA level of AEBV patients' PBMC was (1.3 x 10(6) +/- 1.6 x 10(6)) copies/ml. The average EBV-DNA level of CAEBV infected patients' PBMC was significantly higher than that of AEBV infected patients' PBMC (P<0.01). The cell fractionation and FISH in seven CAEBV patients showed that EBV in CAEBV patients infected not only B cells, but NK cells and CD4+ and CD8+ T cells to different degree, and these patients presented recurrent and persistent infectious mononucleosis (IM)-like symptoms. In 6 CAEBV patients infection mainly occurred to T cells, in one case, infection occurred mainly in CD8+ T cells, and the patient died from fulminant and deadly T lymphocytes proliferative syndrome except presenting firstly high fever, enlargment of the liver, spleen, lymphnode and the severe decrease of one or three kinds of blood cells. In 1 CAEBV patient the infection was mainly found in NK cells, who presented with hypersensitivity to mosquito biting and high IgE level (2500 U/ml). But EBV in seven AEBV patients infection was found only in B cells who presented with only IM for one time and no EBV-infected PBMC were found in the remaining 6 healthy children. There are much more EBV replications and different EBV-infected cell types in CAEBV patients. Detection of EBV-DNA level by real-time fluorescent quantitative PCR and the detection of the type of EBV-infected cells may help in diagnosis, treatment and development evaluation of children with CAEBV infection.

Genomic Imbalances Are Confined to Non-Proliferating Cells in Paediatric Patients with Acute Myeloid Leukaemia and a Normal or Incomplete Karyotype

Leukaemia is often associated with genetic alterations such as translocations, amplifications and deletions, and recurrent chromosome abnormalities are used as markers of diagnostic and prognostic relevance. However, a proportion of acute myeloid leukaemia (AML) cases have an apparently normal karyotype despite comprehensive cytogenetic analysis. Based on conventional cytogenetic analysis of banded chromosomes, we selected a series of 23 paediatric patients with acute myeloid leukaemia and performed whole genome array comparative genome hybridization (aCGH) using DNA samples derived from the same patients. Imbalances involving large chromosomal regions or entire chromosomes were detected by aCGH in seven of the patients studied. Results were validated by fluorescence in situ hybridization (FISH) to both interphase nuclei and metaphase chromosomes using appropriate bacterial artificial chromosome (BAC) probes. The majority of these copy number alterations (CNAs) were confirmed by FISH and found to localize to the interphase rather than metaphase nuclei. Furthermore, the proliferative states of the cells analyzed by FISH were tested by immunofluorescence using an antibody against the proliferation marker pKi67. Interestingly, these experiments showed that, in the vast majority of cases, the changes appeared to be confined to interphase nuclei in a non-proliferative status.

Evaluation of a new cell separator for collection of peripheral blood CD34+ progenitor cells in pediatric patients

This study was conducted to evaluate the performance of the COM.TEC cell separator (Fresenius HemoCare GmbH) for collecting CD34+ cells in pediatric patients who were intended to have autologous peripheral blood progenitor cell transplantation, with respect to collection variables, prediction power of CD34+ cell yield, and influence on donors. A total of 26 pediatric solid tumor patients who received mobilization chemotherapy and granulocyte-colony-stimulating factor underwent CD34+ cell collection (n = 96) using the COM.TEC auto mononuclear cell (MNC) program. Patients were divided into a neuroblastoma (NBL) group and a brain tumor group according to the intensity of prior chemotherapy regimens. The collection variables, cellular variables of leukapheresis products, and the peripheral blood cell counts of patients were compared with those acquired using the COBE Spectra (GambroBCT). The CD34+ cell collection efficiency (CE) and the percentage ratios of actual to predicted CD34+ cell yield indicating prediction power were analyzed. Using the COM.TEC auto MNC program, the processing rate was higher and the product volume was smaller (p < 0.05) than those of the COBE Spectra. Platelet (PLT) reduction in peripheral blood and PLT contamination of the products were significantly lower (p < 0.01). The median CE was less than 60% in both patient groups (50.0 and 48.4%, respectively). The actual collected CD34+ cell yields were medians of 66.9 and 76.1% of the predicted values in NBL group and brain tumor group, respectively. PBPC collections by the COM.TEC cell separator had advantages of high processing rate, low product volume, and low contamination by PLTs of product. Low PLT loss was observed in pediatric patients who need to collect autologous PBPCs. However, applying CD34+ cell yield prediction was not practical for prospective scheduling of the next collection. More specified data need to be accumulated for more accurate prediction of CD34+ cell yield in pediatric patients.

Higher Frequency of Circulating CRTH2 + Natural Killer T Cells in Pediatric Patients with Eosinophilia

Higher Frequency of Circulating CRTH2 + Natural Killer T Cells in Pediatric Patients with Eosinophilia

Immune Reconstitution: How It Should Work, What's Broken, and Why It Matters

Of the multiple cell lineages undergoing reconstitution in the setting of hematopoietic stem cell transplantation (HSCT), this overview will focus on reconstitution of the T cell arm of the immune system. In clinical HSCT, the autologous setting is free of constraints imposed by reactions to allogeneic antigens, but markers are lacking to distinguish reconstitution from transferred cells versus stem or progenitor cells left residual in the host. In allogeneic transplants, the converse is true – the stem cell origins of the reconstituted populations can be determined, but the biology of such reconstitution is confounded by concomitant allogeneic responses. Because of these constraints, the basic biology of T cell immune reconstitution was first delineated in mouse models in which the availability of congenic strains allowed stem cell origins of reconstituted cell populations to be investigated without the presence of confounding allogenic reactions. Two pathways of T cell regeneration were identified: the thymic-dependent maturation of new T cells from marrow progenitors, and thymic-independent expansion of mature peripheral T cells. 1 These initial studies also determined that no other pathways existed by which substantial peripheral populations of T cells were generated, and also established a means for investigating T cell reconstitution in humans by analyzing the expression of isoforms of the leukocyte common antigen CD45 as markers of naive and memory populations which correlated with the pathway of regeneration. They also demonstrated that homeostatic expansion of mature T cells occurred in lymphopenic hosts. Studies of such expansion have formed central concepts of homeostasis – that T cell levels are maintained by a balance between cytokine-consuming cells and homeostatic cytokines that support the maturation, proliferative expansion and survival of T cells.2 In translating these murine findings to humans, the recovery of CD4+ T cells in pediatric patients who were severely lymphopenic following chemotherapy treatment were initially tracked (Figure 1). There was a strong correlation between the recovery of total CD4+ T cell numbers and the recovery of naive, CD45RA+CD45RO- CD4+ T cells after as few as six months. 3 Furthermore, recovery of naive CD4+ cells was associated with a marked expansion of thymus volume, consistent with a dynamic regulation of the thymus.

The levels of CD4+CD25+ regulatory T cells in paediatric patients with allergic rhinitis and bronchial asthma

Our purpose was to determine whether numbers of CD4(+)CD25(+) T [T regulatory (T(reg))] cells and mRNA expression of functional molecules of T(reg) are related to airway allergy and disease severity in 51 paediatric patients with allergic rhinitis or bronchial asthma and 47 healthy controls. Surface markers were evaluated with flow cytometry, and mRNA was determined with real-time polymerase chain reaction. Children with allergic disease had fewer CD4(+)CD25(+) T cells (8 x 49% +/- 2 x 41% versus 9 x 58% +/- 2 x 43%, P<0 x 05) and CD4(+)CD25(hi) T cells (1 x 32% +/- 0 x 68% versus 1 x 70% +/- 0 x 68%, P<0 x 01) than control subjects. Numbers of CD4(+)CD25(+) and CD4(+)CD25(hi) T lymphocytes were higher in children with persistent allergic rhinitis and/or moderate-severe bronchial asthma than in those with respective milder disease. The number of T(reg) cells was correlated positively with total immunoglobulin E level. The mRNA expression of forkhead box P3 (FoxP3) was increased in moderate-severe versus mild asthma (2 x 93 +/- 0 x 38 versus 1 x 60 +/- 0 x 31, P< 0 x 01). Patients with moderate-severe bronchial asthma also had increased mRNA expression of interleukin (IL)-10 compared with patients with mild asthma (15 x 24 +/- 4 x 07 versus 3 x 77 +/- 2 x 18, P<0 x 01). The suppressive function of T(reg) cells from patients with more severe asthma was competent in vitro. On average, decreased numbers of T(reg) cells in children with allergic airway disease might represent a defect of the T(reg) population. With increased expression of FoxP3 and IL-10 in T(reg) from patients with relatively severe allergic disease, adaptive and functional T(reg) might be generated in response to aggravated atopy and disease severity.

Pharmacology of intracellular cytosine-arabinoside-5′-triphosphate in malignant cells of pediatric patients with initial or relapsed leukemia and in normal lymphocytes

The prodrug cytosinearabinoside (ara-C) is widely used in the treatment of acute leukemias. The active drug is the intracellular metabolite cytosine-arabinoside-5'-triphosphate (ara-CTP). The purpose of the present study was to investigate the relation between sensitivity and pharmacokinetic parameters Cmax, t1/2 and AUC of ara-CTP. The obtained results were compared to previous studies. Cmax, t1/2 and AUC of ara-CTP were assessed in leukemic cells of 17 pediatric patients with acute lymphoblastic leukemia (ALL) and in 6 lymphoblastic cell lines and compared with normal lymphocytes of 9 healthy donors by high pressure liquid chromatography (HPLC). The sensitivity of the cells against ara-C was determined by the MTT assay. The intracellular accumulation of ara-CTP was significantly lower in normal lymphocytes (Cmax 47.7-60.9 pmol/10(6) cells) compared to leukemic cell lines (Cmax 11-1128 pmol/10(6) cells) and leukemic cells of our patients (Cmax 85.9-631 pmol/10(6) cells). Similar results were found for the AUC. There was no significant difference between initial and relapsed leukemias in our small cohort. A correlation between sensitivity in terms of IC50 values and the intracellular ara-CTP accumulation was observed in cell lines, but not in leukemic cells and normal lymphocytes from healthy donors. Pharmacokinetic parameters varied tremendously in leukemic cells in contrast to normal lymphocytes without a difference in sensitivity. It is worthwhile to compare literature data to assess an optimal dosage of ara-C in pediatric patients.

Early Recovery of Thymus-Derived Naïve T Cells in Pediatric Patients (pts) Treated with Non-Myeloablative Allogeneic Peripheral Blood Stem Cell Transplantation (NMSCT) for Cancer.

Background: Current SCT approaches consistently achieve rapid donor myeloid engraftment, but delayed immune recovery remains a significant obstacle and results in increased risk of infection and relapse. T cells are regenerated via 2 pathways, thymus-derived and peripheral expansion, processes for which IL-7 is critical. We postulated that non-myeloablative pre-transplant conditioning might preserve thymic function in pediatric SCT recipients thus enhancing thymus-derived naïve T cell regeneration.Methods: We analyzed T cell subsets, T cell receptor excision circles (TREC), and IL-7 levels in peripheral blood after SCT in 21 pediatric pts with high-risk malignancies (median age 14, range 4–21). Fludarabine-based induction chemotherapy was administered for disease control and targeted CD4 count reduction. Pre-transplant conditioning consisted of cyclophosphamide (1,200 mg/m2/day) and fludarabine (30 mg/m2/day) × 4 days plus melphalan (100 mg/m2 × 1 dose in sarcoma pts). Grafts consisted of G-CSF mobilized unmodified peripheral blood stem cells from 5–6/6 HLA-matched first-degree relatives (median CD34 dose 11.7 × 10E6/kg, range 4.4–19.1; median CD3 dose 416 × 10E6/kg, range 228–815). Cyclosporine was used for GVHD prophylaxis.Results: Donor-derived engraftment was rapid (absolute neutrophil count > 500/uL median day 9, range 8–11). Complete donor lymphoid chimerism (>95% by VNTR-PCR on CD3 sorted peripheral blood) was achieved in all by day 28. Immune recovery was brisk and sustained. Substantial numbers of naïve (CD45RA+/CD62L+) CD4+ and CD8+ T-cells were detected at day 28 (Fig 1). There was a steady increase in TREC from 3 to 12 months consistent with early, robust thymic-dependant T cell generation (Fig 2). This was not seen in adult pts treated on a parallel trial (data not shown). IL-7 levels were elevated and inversely correlated with T cell counts (r=−0.56, p<0.0001).Conclusions: Targeted immune depletion and NMSCT results in rapid, sustained immune reconstitution in pediatric pts with malignancy. Preserved thymic function appears to contribute to naïve T cell recovery in this setting. We postulate that non-myeloablative conditioning is thymus sparing and that this, in combination with immune depletion-induced IL-7 elevation, promotes early thymic-derived lymphoid recovery. This approach may serve as a strategy to overcome the prolonged immunodeficiency commonly encountered after allogeneic SCT in pediatrics and might be used as a platform to direct allogeneic anti-tumor immune responses in high-risk childhood cancers. [Display omitted] [Display omitted]

Gallbladder wall inflammatory cells in pediatric patients with biliary dyskinesia and cholelithiasis: a pilot study

Background/Purpose Inflammation has been implicated in functional gastrointestinal disorders, including functional dyspepsia and irritable bowel syndrome. This study was undertaken to evaluate gallbladder wall inflammatory cells in children with abdominal pain related to gallstones and biliary dyskinesia to determine the candidate cell types that may be contributing to the pathophysiology of these entities. Methods Gallbladder specimens from 20 patients with cholelithiasis, 20 biliary patients with dyskinesia, and 12 autopsy controls were evaluated in a blinded fashion. Eosinophil, tryptase-positive, and CD3+ cell densities were determined for the lamina propria and muscularis mucosa layers and compared between groups. Results Patients with biliary dyskinesia and cholelithiasis had a 9- to 12-fold increase in mean and peak mast cell densities, respectively, in both layers as compared with controls. Peak (13.7 vs 8.4) and mean (9.2 vs 5.2) CD3+ cell densities were increased in the muscularis mucosae of cholelithiasis specimens as compared with biliary dyskinesia specimens. Conclusion Gallbladder wall inflammatory cell densities, particularly mast cells, differ between children with cholelithiasis, children with biliary dyskinesia, and controls. Future studies are warranted to define the roles for specific inflammatory cell types.

The Correlation between G-CSF Dosage and the Number of Collected CD34+ Cells in Pediatric Patients Undergoing Peripheral Blood Stem Cell Mobilization

Background: In high risk malignancies of pediatric patients, many investigators have explored the use of stronger myeloablative regimens with autologous peripheral blood stem cell rescue such as the Tandem protocol. As the collection of peripheral blood stem cells in children have many risks, it is important to achieve the maximum number of CD34+ cells per leukapheresis. We analyzed the use of different doses of granulocyte-colony stimulating factor during a mobilization for an increased number of CD34+ cells. Methods: A retrospective chart review was performed for 31 patients undergoing autologous peripheral blood stem cell mobilization. All patients had received specific chemotherapies. At the nadir, each patient was injected with 5 μg/kg/day G-CSF. When the level of white blood cells reached more than 1,000/μL, patients were divided according to the different dose of G-CSF that was given per day: Group I, 5μg/kg, Group II, 10μg/kg, Group III, 15μg/kg. After 2~3 days, leukapheresis was performed, and then the number of CD34+ cells and other cells were counted. Results: In group III, the number of collected CD34+ cells was 19.86±14.45×10 6 /kg and significantly higher than in the other two groups. Also, group III had significantly higher numbers of total nucleated cell, mononucleated cells, white blood cells, though the absolute neutrophil count compared with the other groups. There were no serious adverse effects associated with the higher G-CSF doses employed. Conclusion: At nadir, a pediatric patient received 5 μg/kg G-CSF daily until the WBC count reached more than 1,000/μL. If the patient is injected with 15μg/kg/day of G-CSF, we can achieve a more sufficient CD34+ cell yield with one leukapheresis, safely. (Korean J Hematol 2006;41:243-249.)

Collection of peripheral progenitor cells in paediatric patients with a new programme for the collection of mononuclear cells.

When harvesting peripheral progenitor cells (PPC) in children, the special situation of their circulatory system has to be taken into account. Therefore, extracorporeal blood volume and product volume should be small to avoid side effects. Nine children (age 2-14 years, weight 12.8-58.5 kg) with malignancies underwent 10 PPC collections with the MNC programme of the Amicus blood cell separator. The disposable kit was primed with red blood cells (RBCs) or human albumin to avoid circulatory side effects. The children were monitored for blood pressure and heart rate during the whole apheresis procedure. A median blood volume of 4,577 ml (range 3,536-8,596 ml) was processed in a separation time of 270 min (range 176-331 min). The median product weight was 81 g (range 53-107 g) and the yield of CD 34 antigen expressing cells was 12.5 x 10(6)/kg body weight (range 1.8-26 x 10(6)/kg body weight). Only one child had to undergo a second apheresis to collect the desired transplantation dose. The median platelet contamination of the product was 0.32 x 10(11) (0.13-0.85 x 10(11)). No circulatory side effects were observed. Blood flow alarms occurred in seven of ten aphereses and one collection had to be terminated due to insufficient flow. PPC can be efficiently collected in children with the MNC programme without circulatory side effects. The platelet contamination of the product was low due to the elutriation principle of the collection process, thereby avoiding thrombocytopenic bleeding episodes in the patients.

SIALYL-Tn ANTIGEN DISTRIBUTION IN HELICOBACTER PYLORI CHRONIC GASTRITIS IN CHILDREN: AN IMMUNOHISTOCHEMICAL STUDY

Sialyl-Tn antigen (STn) is a mucin-type carbohydrate normally present in goblet cells of small and large bowel. STn expression has been demonstrated to occur in complete and incomplete intestinal metaplasia as well as in many carcinomas but in no normal gastric cell. The aim of our present study was to evaluate the distribution of STn in Helicobacter pylori chronic gastritis (HpCG) of pediatric patients. Eighteen gastric biopsies from 15 children (mean age: 11.5 years) with HpCG, 9 gastric biopsies from 9 children without H. pylori infection, and 1 heterotopic gastric mucosa in Meckel's diverticulum were immunostained using the anti-STn antibody STn1 (18/18), NCL-MUC-1 (7/18), and NCL-MUC-2 (18/18) antibodies. Also, sulfated mucosubstances were investigated with the Alcian Blue-Periodic Acid Schiff (AB-PAS), pH 1.0 stain. Although with different intensity (weak in 5/18, moderate 9/18, and intense 4/18) all cases with HpCG exhibited STn immunoreactivity. The expression of STn was found to be located mainly to the supranuclear region of the epithelial cells at the foveolae and glandular necks, with occasional cells showing diffuse cytoplasmic staining. When reactivity was intense, it was for the most part found in the cells at the neck of the glands. The mucus out of the luminal border above the positive cells was usually also stained. MUC-1 was negative (2/7) or weakly positive (5/7) in a few surface mucous cells. MUC-2 was negative (16/18) or occasionally detected in some foveolar and surface cells (2/18). AB-PAS pH 1.0 revealed the presence of sulfomucins in the cytoplasm of isolated cells of gastric pits and glands of most cases (11/15). None of these findings was observed in the control group. We conclude that STn can be identified in gastric cells of pediatric patients with HpCG and that this does not correlate with other mucosubtances markers. The findings could indicate that minimal intestinal metaplasia takes place in children with HpCG.

Colledction of Peripheral Progenitor Cells in Pediatric Patients with Reduced Volume Technique

Background: When peripheral progenitor cells (PPC) are harvested in children the special situation of their circulatory system has to be taken into account. Extracorporeal blood volume and product volume should be low to avoid side effects. Setting: Hemapheresis unit and pediatric oncologic ward of a University Clinic. Patients: 12 children with neuroblastoma, Ewing sarcoma, unclassified malignant bone tumor, medulloblastoma, pheochromocytoma, rhabdomyosarcoma and Wilms tumor. Material and Methods: The children underwent 24 PPC collections with a continuous flow system and a reduced volume programme. PPC were mobilized by chemotherapy and G-CSF application. Blood counts and CD34 antigen-expressing cells were determined before apheresis and in the PPC product. PPC were frozen at controlled rates and stored in liquid nitrogen until they were used for autologous transplantation. Results: Except for anticoagulant-induced venous irritation, no adverse side effects were observed. The median product volume was 59 ml (range 34–119 ml) and the yield of CD34 antigen-expressing cells was 1.9 × 10⁶/kg body weight (0.1 × 10⁶– 20.4 × 10⁶/kg). The platelet contamination of the products was 0.25 × 10¹¹(0.05 × 10¹¹–1.43 × 10¹¹). Median hematopoietic reconstitution for platelets was achieved on day 27 after transplantation and for leukocytes on day 10.5, respectively. Conclusion: PPC can be easily collected in children with the reduced product volume technique. The system is adopted to the small blood volume of infants so that no circulatory side effects were observed. The reduced volume technique helps lessening the quantity of dimethylsulfoxide needed for cryopreservation as well as reducing storage space. Platelet contamination of the product was low due to retransfusion of platelet-rich plasma to the patients prior to PPC harvesting.

Evaluation of immune status in pediatric recipients of hepatic orthografts

Survival in children following hepatic transplantation is better than in adults. We evaluated the immunologic status of both pediatric and adult patients prior to and following transplantation, to determine if there were differences in immunologic status that might contribute to this phenomenon. Studies included determination of mononuclear cell subsets, including T cells, T helper cells, T suppressor cells, and monocytes. In addition, we evaluated immune function by studying in vitro immunoglobulin (Ig) synthesis. Following transplantation, the T8 (cytotoxic/suppressor) cells in pediatric patients did not decline, whereas in adults they did (P less than .05). In vitro Ig synthesis in both adult and pediatric patients declined following transplantation. By 4 weeks, the adult group had begun to recover, whereas in the pediatric group there was still significant suppression (P less than .05). These studies suggest that there is decreased immune function in children who are recipients of hepatic allografts. Such findings could contribute to better outcome of hepatic allografting in children.