Immune thrombocytopenic purpura in children.

Immune thrombocytopenic purpura (ITP) is the most commonly acquired bleeding illness in children. 70-80% of children resolve from acute state within few weeks and months with a complete retrospective changes in recovery of reference values. The aim of this work was to evaluate a group of patients with diagnosis of ITP treated or followed from 1979 to 1999. There were found no differences in achieving the remission in the acute phase of ITP (this means till the 6th month from the first documented thrombocytopenia) according to retrospective analysis and comparison of three groups of children in whom the diagnosis of ITP was made from 1979 to 1991 (group A), from 1992 to 1994 (group B) and from 1995 to 1999 (group C). The groups differed in their therapeutic strategies in various time periods as to the time of the diagnosis. In group A, 75% of patients were treated with oral corticosteroids (prednisone). In group B, 10.8% of patients were treated with i.v. application of corticosteroids and 43.3% had no therapy in the acute phase. In group C, 28% of patients were treated with i.v. application of corticosteroids and 36.6% of patients had no therapy applied. A comparable degree of remission in the acute phase with 48%, 54%, or 50% of children with reference values of platelets at the time of six months from the beginning of the disease were achieved. In the group A, the remission was achieved in 85% of children at the end of the 5th year of the follow-up, in the group B in 75% of children to the latest control of platelets in our outcome clinic for children and follow up in the group C in 68% of children the remission was achieved after one year. (Tab. 4, Fig. 2, Ref. 10.)

Interleukin-1 receptors and receptor antagonist in haemodialysis.

BACKGROUND: Biological activity of interleukin-1 (IL-1) depends on the number and type of IL-1 receptors on target cells and on the amounts of its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1ra). METHODS: Expression of IL-1 receptor was studied on the peripheral blood mononuclear cells of 20 end-stage renal-disease patients maintained by chronic haemodialysis by means of either polysulphone (10 patients) or cuprophane membranes (10 patients) and compared to that of normal controls. Plasma and cellular levels of IL-1ra and IL-1 beta were also measured. RESULTS: The proportion of monocytes expressing the IL-1 receptor was strikingly higher in haemodialysis patients than in the healthy population. This proportion further increased during haemodialysis with cuprophane but not with polysulphone. Expression of the IL-1 receptor on lymphocytes was very low in both controls and dialysed patients; in the latter there was no intradialytic variation. Plasma concentrations of IL-1 beta and IL-1ra were elevated in haemodialysis patients and undetectable in controls. Whereas plasma IL-1 beta decreased throughout haemodialysis, IL-1ra further increased, with no significant differences between the two membranes used. Total cellular IL-1 beta and IL-1ra were also higher in the patient group than in the healthy controls. A further increase of both IL-1 beta and IL-1ra was detected at the end of the haemodialysis session with any membrane. CONCLUSIONS: Monocytes of haemodialysis patients circulate in a state of activation, which makes them both producer and target of IL-1. Thus there is an autocrine upregulation of IL-1 production. Although IL-1ra levels are high, they are most likely to be expression of monocyte activation rather than represent effective inhibitors of IL-1 activity.

Multicenter randomized study of two once daily regimens in the initial management of community-acquired respiratory tract infections in 163 children: azithromycin versus ceftibuten.

In a randomized trial, we compared the efficacy and toxicity of azithromycin and ceftibuten once daily in the initial (empiric) therapy of proven or suspected community-acquired respiratory tract infections (CARTI) in 163 pediatric patients: 95.5% of those treated with azithromycin and 83.6% of those treated with ceftibuten were cured or improved. Streptococcus pneumoniae was more frequently eradicated in the azithromycin than in the ceftibuten group, whereas gram-negative bacilli were more susceptible to ceftibuten. Elimination rates for Staphylococcus aureus and Haemophilus influenzae were similar; adverse reactions did not differ in both arms. Thus, azithromycin was more effective but equally safe than ceftibuten in the initial therapy of pediatric CARTI.

Direct inhibition of human CD8+ lymphocyte activation by cyclosporine A and Rapamune-Sirolimus.

Cyclosporin A (CsA) and Rapamune-Sirolimus (RAP) have been shown to inhibit the in vitro activation of heterogeneous lymphocytes populations, but little is known about their direct actions on isolated CD8+ lymphocytes. In this study the direct effects of RAP and CsA on a highly purified population of CD8+ lymphocytes were examined. Human CD8+ lymphocytes were purified to near homogeneity and stimulated with anti-CD3 antibody, OKT3, or allogeneic cells in the presence of exogenous human recombinant interleukin 2 (IL2). The effects of CsA and RAP on cell proliferation, the entry into the S phase of the cell cycle, the surface expression of Tac antigen, and the release of soluble IL2 receptor and soluble CD8 were analyzed. When CsA and RAP were included in the stimulated CD8+ lymphocyte cultures, these responses were inhibited. OKT3-stimulated CD8+ lymphocytes were sensitive to lower concentrations of the immunosuppressants than those previously reported for peripheral blood mononuclear cells. RAP was effective at a lower dose than CsA and when the agents were applied in combination, cell proliferation was synergistically inhibited. These results demonstrate that CsA and RAP can inhibit the activation and proliferation of purified CD8+ lymphocytes in response to OKT3 or alloantigen in the presence of IL2.

Comparison of flow and image cytometry for DNA content analysis of fresh and formalin-fixed, paraffin-embedded tissue in breast carcinoma.

DNA ploidy and S-phase fraction are considered to be prognostic variables in breast carcinoma. DNA content of 35 cases of breast carcinoma of varying histologic types and nuclear grades was analyzed by flow cytometry and image analysis in both fresh and formalin-fixed, paraffin-embedded tissue. Fresh cell and deparaffinized nuclear suspensions were used for flow cytometry. Fresh and deparaffinized tumor tissue samples were used for image analysis. The results of analysis for DNA ploidy, DNA index of DNA aneuploid Go/G1 peaks, and S-phase fraction were compared in different tissue preparations for both techniques. The two techniques produced comparable DNA ploidy results with both fresh and formalin-fixed, paraffin-embedded tissue. Sensitivity for detection of DNA aneuploidy was somewhat greater by image analysis, particularly in deparaffinized tissue. There was 89% agreement in detection of DNA aneuploidy by flow cytometry in fresh and paraffin-embedded, formalin-fixed tissue; the coefficients of variation of the DNA diploid Go/G1 peaks were much wider in the latter. In image analysis there was 91% agreement between fresh and fixed specimens. Agreement between the flow cytometry and image analysis in fresh specimens was 91%; in deparaffinized nuclear suspensions it was 94%. There is a high degree of correlation between the values of DNA index of DNA aneuploid Go/G1 peaks; the estimates of S-phase fraction are much more variable. Results also show a good correlation of the DNA ploidy with the nuclear grades.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cyclosporine and rapamycin on immunoglobulin production by preactivated human B cells.

In order to assess the direct effects of cyclosporine A (CsA) and rapamycin on B cells, we utilized a two-segment culture system of highly purified B lymphocytes consisting of induction (activation) in the presence of the formalinized Staphylococcus aureus bacteria and IL-2, and differentiation, respectively, in the presence of various combinations of cytokines. Results show that rapamycin strongly inhibited production of both IgM and IgG measured at the end of the secondary culture supported by IL-2/IL-6, whereas CsA up-regulated the immunoglobulin production. The stimulatory effect of CsA was also observed when preactivated B cells were recultured in absence of any cytokines. These results show that rapamycin and CsA have clearly distinct effects on human B lymphocyte responses in vitro. Rapamycin is a more potent in vitro immunosuppressant of B lymphocytes than CsA. It is effective at significantly lower concentrations, and it does not stimulate either the proliferation or antibody production by preactivated B cells.

Identification of functional domains of adenovirus tumor-specific transplantation antigen in types 5 and 12 by viable viruses carrying chimeric E1A genes.

The adenovirus (Ad) E1A gene induces in immunized animals a strong tumor transplantation (TSTA) immunity against Ad tumors. Such immunity with group-A and group-C viruses is highly group-specific and no cross-protection is detected between serotypes 5 and 12. This fact was used to map the domains of the Ad5 and Ad12 E1A gene products, respectively, which control the TSTA. We constructed a library of 8 recombinant viruses (H5sub1101 through H5sub1108) which carry chimeric Ad5/Ad12 E1A genes in the background of Ad5. The chimeric genes are functional and these viruses are viable. Some of these constructs induce strong and highly specific tumor syngraft immunity in immunized rats. The viruses carrying the 5' terminus of the first E1A exon derived from Ad12 (viruses H5sub1101, H5sub1102 and H5sub1103) induce strong protection against Ad12 tumors irrespective of the rest of their E1A sequence. The viruses which carry the second exon of the Ad5 E1A gene (viruses H5sub1101, H5sub1102 and H5sub1106) protect against group-C tumors, regardless of the origin of the rest of their E1A gene. The 2 viruses that carry the 5' E1A terminus of the first exon of Ad12 and the second exon of Ad5 (H5sub1101 and H5sub1102) are thus effective in inducing immunity against Ad12 tumors as well as against Ad2 tumors. The viruses which carry the 5' terminus of the first exon derived from Ad5 and the second exon of Ad12 (H5sub1107 and H5sub 1108) fail to induce immunity against either tumor. Expression of only the truncated 5' terminus of the Ad12 E1A gene (viruses H5sub1104 and H5sub1105) is sufficient for induction of Ad12 TSTA. Our results provide direct and unequivocal in vivo evidence that TSTA activities of adenovirus groups A and C are controlled by different domains of their respective E1A genes. The Ad12 TSTA is a function of the 5' terminus of the first E1A exon, while the Ad5 TSTA is coded for by the 3' exon of its E1A gene.

IL-4 receptor expression by SAC-activated B-lymphocytes: its role in B-cell proliferation and the effect of cyclosporine (CsA), prednisolone and verapamil.

Expression of the IL-4 receptor was studied in a highly purified population of human B-lymphocytes stimulated by Staphylococcus aureus, cowan I (SAC). Flow cytometric analysis showed that incubation with SAC in the absence of detectable levels of IL-2, IL-4 and IL-6 resulted in a striking increase in cellular binding of IL-4. The SAC-stimulated B-cells responded to exogenous IL-4 by DNA synthesis. This response was unaffected by CsA or prednisolone, but was inhibited by the Ca2+ channel blocker verapamil.

Responsiveness of preactivated B cells to IL-2 and IL-6. Effect of cyclosporine and rapamycin.

In order to determine which drug may be more effective in clinical abnormalities associated with polyclonal B lymphocyte activation, we compared the in vitro effects of CsA and rapamycin on proliferation or differentiation of preactivated B cells. For that purpose, highly purified B lymphocytes were preactivated in the presence of formalinized Staphylococcus aureus bacteria and then recultured in the presence or in the absence of either rIL-2, rIL-6, or combination or rIL-2 and rIL-6. After 48 hr in culture, S. aureus bacteria upregulated significantly the binding of phycoerythrin-conjugated IL-2 and IL-6, respectively, by purified B lymphocytes, indicating generation and/or upregulation of receptors for these cytokines. Such preactivated B lymphocytes proliferated in response to optimal concentrations of rIL-2, whereas the addition of rIL-6 to preactivated cells was always accompanied by a decrease of the proliferation rate. CsA upregulated cell proliferation when it was added in the second culture period in the presence or in the absence of rIL-6, whereas rapamycin had no effect in these cases. A combination of rIL-2 plus rIL-6 upregulated significantly the proliferative responses of preactivated B cells. In such cultures both CsA and rapamycin had an inhibitory effect on the proliferative responses. IgM production was unaffected by the addition of rIL-6 to cultures of preactivated B cells, whereas addition of rIL-2 and of the IL-2/IL-6 combination enhanced considerably IgM production. Irrespective of cytokines added, CsA upregulated the production of IgM. In contrast, rapamycin inhibited IgM production in all cases. Our results indicate that, in this experimental system, rapamycin is an effective immunosuppressive agent and its use, at least in vitro, is not accompanied by an upregulation of either the proliferation or differentiation of B lymphocytes.

Significance of increased proportion of CD10-positive cells in nonmalignant bone marrows of children.

An immunophenotypic pattern characterized by increased proportions of CD10, CD19, and HLA-DR+ cells is observed in the bone marrow of some children with nonmalignant hematologic diagnoses. The purpose of this study was to ascertain the clinical and pathologic significance of this immunophenotype. The morphologic and immunophenotypic results of bone marrow specimens from 23 children with nonmalignant hematologic conditions are presented. In 11 children, an increased percentage of immature B-cell precursors was observed, suggesting the presence of B-lineage malignancy. None of the children have developed malignant lymphoproliferative disease in as long as 4 years of follow-up, despite the demonstration of a clonal lymphocyte proliferation in one patient. Although the biologic significance of the bone marrow immunophenotype is not yet understood, it is important to recognize that this lymphocyte pattern occurs commonly in children with nonmalignant hematologic conditions, and may reflect an increase in normally occurring lymphoid precursor cells.

Effect of verapamil on the IL-2 binding to its active receptor and on the release of IL-2 receptor by activated PBMC.

In the present study we have examined the effect of a Ca2+ channel blocker (verapamil) on the binding of IL-2 to its biologically active receptor (IL-2R), as well as on the release of its soluble form (sIL-2R) by peripheral blood mononuclear cells (PBMC) stimulated by a variety of stimuli. In the same culture systems, cyclosporine A (CsA) was also used as an additional dissecting tool. PHA and the Ca2+ ionophore A23187 enhanced both the percentage of PBMC binding phycoerythrin-conjugated IL-2 (PE-IL-2) and the mean fluorescence intensity of this binding. A phorbol-ester (PMA), on the other hand, enhanced only slightly the proportion of PE-IL-2 binding cells. The two stimulatory combinations (PHA/PMA and A23187/PMA) also up-regulated the proportion of PE-IL-2 binding cells and the fluorescence intensity; the PHA/PMA combination was the most potent of all stimuli used. These two stimulatory combinations, and PHA alone, were also associated with maximal in vitro release of sIL-2R. Verapamil significantly down-regulated PE-IL-2 binding in all culture systems and it convincingly inhibited the release of sIL-2R. Furthermore, this mode of action of verapamil was concentration-dependent. CsA, on the other hand, inhibited the binding of PE-IL-2 to all stimulant-activated PBMC and had only a slight inhibitory effect on the in vitro release of sIL-2R. Our results indicate that there is a correlation between the binding of IL-2 to biologically active receptors on the surface of stimulant-activated PBMC and the release of the soluble form of IL-2R by the same cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Uremic serum effects on peripheral blood mononuclear cell and purified T lymphocyte responses.

We have studied the effects of uremic serum on the activation state and function of normal lymphocytes in vitro, by examining both accessory cell-dependent and accessory cell-independent responses. Uremic serum was obtained from patients on conservative treatment and from the same patients after they have undergone six months of maintenance hemodialysis. Uremic serum inhibited the proliferative responses to mitogens and to recombinant IL-2 (rIL-2) of both peripheral blood mononuclear cells (PBMC) and purified T cell populations. However, the responsiveness to IL-2 of pre-formed lymphoblasts, obtained from both PBMC and purified T cells, in the presence of uremic serum was similar to that obtained in the presence of normal serum, or was even enhanced. Uremic serum did not affect the cellular IL-2 receptor alpha (IL-2R) generation though it inhibited significantly the release of soluble IL-2 receptor (sIL-2R) and the production of IL-2 after mitogenic stimulation. Uremic serum from patients after six months of hemodialysis enhanced, but did not completely restore, proliferative responses and IL-2 production by control PBMC. Neither IL-1 nor IL-2R, which are present at elevated concentrations in uremic serum, appeared to be responsible for serum effects on in vitro responses of control lymphocytes. In conclusion, our results indicate that uremic serum affects both accessory cell-mediated and accessory cell-independent normal T cell responses. Uremic serum inhibition of T cell proliferation is associated with down-regulation of IL-2 synthesis by lymphocytes and the induction of an abnormal state of activation of lymphoblasts which is further enhanced following chronic hemodialysis.

Binding of phycoerythrin-conjugated interleukin-6 to in vitro-activated human peripheral blood mononuclear cells--effect of immunosuppressive agents and of a calcium channel blocker.

We studied the effect of cyclosporine A, prednisolone, and the Ca2+ channel blocker verapamil on interleukin-6 binding to mitogen-activated peripheral blood mononuclear cells, using a flow cytometric technique and phycoerythrin-conjugated IL-6. All mitogenic stimuli up-regulated IL-6 binding to a variable degree. PHA alone or in combination with PMA was the most effective stimulant in up-regulating IL-6 binding in all the experiments performed. The main changes in IL-6 binding were seen in the large cell cluster, which consisted mainly of lymphoblasts. PHA and PHA/PMA, however, also up-regulated the mean fluorescence intensity on the small cell cluster, which consisted mainly of quiescent lymphocytes. The overall effect of the three pharmacological agents on mitogen-up-regulated IL-6 binding was minimal; most significant were a down-regulation by all three agents of IL-6 binding by small lymphocytes in PHA/PMA cultures, a down-regulation of IL-6 binding by CsA in PHA/PMA-induced large PBMC, and an up-regulation by verapamil of PMA-induced IL-6 binding in large PBMC. Measurements of IL-2 binding and of IL-6 production in the same cultures showed a different pattern than that seen with IL-6 binding, as well as different CsA, prednisolone, and verapamil action. In conclusion, by using a new flow cytometric technique providing information both about the quantity of bound cytokine and about the proportion of IL-6-binding cells, we have demonstrated that IL-6 receptor expression in vitro by PBMC can be up-regulated by the use of stimulants differing in the signal transduction pathways they activate. In addition, by using different pharmacological agents and stimuli to dissect different activation pathways of the in vitro immune response, we conclude that IL-6R generation is regulated differently from IL-6 production. Furthermore, since CsA and prednisolone are known inhibitors of in vitro IL-2 production, our results indicate that IL-6R generation does not rely exclusively on the presence of IL-2.

IL-2 responsiveness of lectin-induced lymphoblasts: soluble IL-2 receptor release and differential in vitro effects of immunosuppressants.

We examined the mode of action of different immunosuppressants on the responsiveness of phytohemagglutinin (PHA)-induced lymphoblasts further stimulated by recombinant interleukin-2 (rIL-2). The stimulation of PHA blasts with rIL-2 resulted in an enhancement of tritiated thymidine ([3H]TdR) incorporation and of soluble interleukin-2 receptor (sIL-2R) release. Cyclosporin A (CsA) and prednisolone inhibited in different ways the responsiveness of PHA pre-stimulated blood mononuclear cells (PBMC) to rIL-2, as measured by [3H]TdR incorporation. The addition of CsA resulted in considerable enhancement of the release of sIL-2R, whereas the addition of prednisolone was associated with a similar enhancement only when the higher concentrations of rIL-2 were employed. EGTA, a calcium (Ca2+) chelator, and verapamil, a Ca2+ channel blocker, inhibited [3H]TdR incorporation in a concentration-dependent manner. EGTA inhibited sIL-2R release in the same manner when used alone, and reversed the CsA- and prednisolone-induced enhancement of sIL-2R release by rIL-2 induced lymphoblasts, when used in combination with CsA or prednisolone. Verapamil had a similar but less striking effect. The effects of CsA and prednisolone were also studied in PHA-induced blasts originating from purified CD4+ or CD8+ lymphocytes. Stimulation of these blasts with rIL-2 resulted in higher [3H]TdR incorporation by CD8+ blasts than by CD4+ blasts: however, no sIL-2R release was detected in supernatants of either CD4+ or of CD8+ blasts. Both CsA and prednisolone inhibited the rIL-2-induced enhancement of [3H]TdR incorporation by both T-cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hemodialysis on activation of lymphocytes: analysis by an in vitro dialysis model.

Patients undergoing maintenance hemodialysis have impaired immune responsiveness, which appears to deteriorate progressively with the duration of the replacement treatment. It has been suggested that it is caused by a chronic preactivation state of T cells caused by hemodialysis. Each treatment session has been compared with a recurring "acute-phase" inflammatory reaction. In this study, the acute effects of hemodialysis on the activation state and functional responsiveness of normal peripheral blood mononuclear cells have been evaluated by use of an in vitro dialysis model. The dialysis was carried out with either cuprophan or polysulfone membranes, with or without sodium acetate in the dialysis fluid. It was observed that a single session of in vitro dialysis did not induce production of interleukin-2 (IL-2) and did not alter the expression of IL-2 receptor (IL-2R) on the cytoplasmic membrane and the secretion of soluble IL-2R, whereas it induced transcription of mRNA for IL-2R. The proliferative response of lymphocytes to phytohemagglutinin or IL-2 in vitro also did not change after a single dialysis session. There was only a slight decrease of the release of soluble IL-2 receptor by phytohemagglutinin-stimulated cells after dialysis. Dialysis induced an active synthesis of IL-1 by peripheral blood mononuclear cells, even in the absence of sodium acetate in the dialysate bath, but there was no release of IL-1 to the circulating medium. The results show that a single dialysis encounter can acutely prime the activation of human peripheral blood mononuclear cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Teaching pathology without lectures through computer-based exercises, small-group discussions and reading.

The Department of Pathology at Robert Wood Johnson Medical School teaches a 205-hour, one semester course to 150 second-year students in the fall semester of the second year. The course begins with General Pathology (4 weeks) and continues with Systemic Pathology (12 weeks). In 1986, lectures were eliminated as a means of conveying the course content. Students were expected to read the textbook and to participate in obligatory small group discussions. Extensive computer based self-learning materials were provided in both MS/DOS and Macintosh formats. In 1991, these computer resources included the full text of Robbins Pathologic Basis of Disease, extensive question banks and a guide through the Slice of Life Videodisc. Over the past 6 years this modified curriculum has been enthusiastically received by the students and faculty. Student performance on internal and external examinations has been excellent. Approximately 4% of the graduating students select pathology for their post-graduate training.