Leukemic and non-leukemic lymphocytes from patients with Li Fraumeni syndrome demonstrate loss of p53 function,Bcl-2 family dysregulation and intrinsic resistance to conventional chemotherapeutic drugs but not flavopiridol

Li Fraumeni syndrome (LFS) is characterised by a predisposition to the early onset of certain tumors and is associated with germline mutation of the anti-oncogene p53. In this study we analysed the in vitro responses of lymphocytes from two LFS patients to chemotherapeutic drugs in terms of apoptosis induction and the expression of key intracellular proteins that regulate this process. One of the LFS patients also suffered from B-cell chronic lymphocytic leukemia (B-CLL) and hence presented with a light-chain restricted B-cell lymphocytosis while the other patient had entirely normal blood counts. The B-lymphocytes from both LFS patients showed a marked degree of resistance to chlorambucil and fludarabine when compared to age-matched controls but were remarkably sensitive to the novel flavone, flavopiridol. Loss of function of p53 was demonstrated by a failure to induce Bax and p21 protein expression. In addition, altered basal expression patterns of Bcl-2 and Bax, two key regulators of apoptosis, were found in the LFS lymphocytes when compared with controls. These results suggest that LFS lymphocytes carrying a p53 mutation show intrinsic resistance to conventional chemotherapeutic drugs and this is associated with dysregulation of Bcl-2 family proteins. Furthermore, The innate resistance profile was similar in leukemic and non-leukemic lymphocytes and was therefore independent of genetic changes acquired during malignant transformation. Novel agents that induce p53-independent cell killing may be useful not only in the treatment of LFS-associated tumors but also drug resistant tumors in general where p53 and/or Bcl-2 family dysregulation is a feature.     

Effect of Beta-Adrenergic Stimulation on the Circulatory Lymphocyte Count: Studies in Normals and in Patients with Chronic Airflow Obstruction

In eight non-allergic patients with chronic airflow obstruction (CAO) and eight age and sex matched, healthy control subjects the circadian variation in circulatory lymphocyte count was studied in relation to serum Cortisol and urinary epinephrine levels. In addition, we investigated the effect of the beta-adrenergic agent terbutaline on the lymphocyte count in two ways: as a long-term effect after 8 days of oral slow-release terbutaline with constant diurnal and nocturnal serum levels in patients, and as a short-term effect by a constant rate infusion of 0.2μg/min over 4hr in normals. Both patients and controls showed similar circadian patterns of urinary epinephrine excretion and lymphocyte counts. Patients with CAO, however, had significantly lower epinephrine levels and significantly higher lymphocyte counts at all hours of observation (every 4 hr from 0800 to 0800 hr the next day), as compared with normal controls. After 8 days of slow-release terbutaline the lymphocyte count in the patient group decreased to levels not significantly different from that of normals. The circadian rhythm of the lymphocytes, however, persisted under terbutaline therapy. No correlation existed between the lymphocyte count modulating factor, serum Cortisol and the lymphocyte count over 24 hr. On placebo infusion in the control persons an increase of lymphocytes over 4hr occurred, as a consequence of circadian rhythmicity. On terbutaline infusion a significant increase of lymphocytes after 1hr was follwed by a decrease to levels significantly below those on the placebo day. The same pattern was found in the leucocyte count. From this study it is concluded that beta-adrenergic stimulation corrects the relative lymphocytosis to counts comparable with normals. Other coinciding factors must regulate, however, the circadian rhythmicity.     

The pattern of peripheral blood leucocyte changes in mice infected with Nematospiroides dubius

Experiments were carried out to define the haematological changes taking place during the first six weeks of a primary infection with Nematospiroides dubius. The general pattern of changes was observed to comprise a rapid increase in circulating leucocytes (4 to 5-fold increase) which consisted of a neutropl a, lymphocytosis, monocytosis and an eosinophilia. However, in strong responder NIH mice leucocyte counts returned to normal more rapidly than in other strains (by day 28). In contrast, in weak responder C57BL/10 mice the leucocyte counts whilst falling significantly relative to day 7 did not return to normal within the experimental period. Mice infected with irradiated larvae did not experience as high a leucocytosis as did mice given an identical number of normal larvae. The peak lymphocytosis, neutrophilia and monocytosis were all lower. The removal of adult worms from infected animals by treatment with pyrantel on days 9, 11, 13 and 16, also significantly altered the pattern of leucocytosis. The neutrophilia which was evident on day 7 returned rapidly to normal, whereas in mice which had retained their worms a peak neutrophilia was observed on day 14. These haematological changes were discussed and related to the failure of host-protective immunity to operate effectively during the early stages of a primary infection with N. dubius.     

Effect of Beta-Adrenergic Stimulation on the Circulatory Lymphocyte Count: Studies in Normals and in Patients with Chronic Airflow Obstruction

In eight non-allergic patients with chronic airflow obstruction (CAO) and eight age and sex matched, healthy control subjects the circadian variation in circulatory lymphocyte count was studied in relation to serum Cortisol and urinary epinephrine levels. In addition, we investigated the effect of the beta-adrenergic agent terbutaline on the lymphocyte count in two ways: as a long-term effect after 8 days of oral slow-release terbutaline with constant diurnal and nocturnal serum levels in patients, and as a short-term effect by a constant rate infusion of 0.2μg/min over 4hr in normals. Both patients and controls showed similar circadian patterns of urinary epinephrine excretion and lymphocyte counts. Patients with CAO, however, had significantly lower epinephrine levels and significantly higher lymphocyte counts at all hours of observation (every 4 hr from 0800 to 0800 hr the next day), as compared with normal controls. After 8 days of slow-release terbutaline the lymphocyte count in the patient group decreased to levels not significantly different from that of normals. The circadian rhythm of the lymphocytes, however, persisted under terbutaline therapy. No correlation existed between the lymphocyte count modulating factor, serum Cortisol and the lymphocyte count over 24 hr. On placebo infusion in the control persons an increase of lymphocytes over 4hr occurred, as a consequence of circadian rhythmicity. On terbutaline infusion a significant increase of lymphocytes after 1hr was follwed by a decrease to levels significantly below those on the placebo day. The same pattern was found in the leucocyte count. From this study it is concluded that beta-adrenergic stimulation corrects the relative lymphocytosis to counts comparable with normals. Other coinciding factors must regulate, however, the circadian rhythmicity.     

Interleukin-12 p40 mRNA Expression in Bovine Leukemia Virus-Infected Animals: Increase in Alymphocytosis but Decrease in Persistent Lymphocytosis

Interleukin-12 (IL-12), a key cytokine in immune regulation, has an important role in activating the cell-mediated immune response in infectious diseases. Recently, a dichotomy between IL-12 and IL-10 regarding progression of a variety diseases has emerged. IL-12 activates type 1 cytokine production and has an antagonistic effect on type 2 cytokines. Here, by using quantitative competitive PCR, we show that peripheral blood mononuclear cells from bovine leukemia virus-infected animals in the alymphocytotic stage of disease express an increased amount of IL-12 p40 mRNA. In contrast, IL-12 p40 mRNA expression by cells from animals with late-stage disease, termed persistent lymphocytosis, was significantly decreased compared to that by normal and alymphocytotic animals. Interestingly, IL-12 p40 mRNA was also detected in tumor-bearing animals. IL-12 p40 expression occurred only in monocytes/macrophages, not B or T lymphocytes. The present study combined with previous findings suggest that IL-12 in bovine leukemia virus-infected animals may regulate production of other cytokines such as gamma interferon and IL-10 and the progression of bovine leukosis in animals that develop more advanced disease such as a persistent lymphocytosis of B cells or B-cell lymphosarcoma.     

Blood and inflammatory cells of the lungfish Lepidosiren paradoxa

A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In summary, this lungfish appears to possess the typical inflammatory granulocytes of teleosts, however, further functional studies are necessary to better understand the polymorphonuclear agranulocyte.     

BOLD钉治疗桡骨小头骨折的临床疗效分析

目的:报告BOLD钉治疗Mason II型、未累及桡骨颈的Mason III型桡骨小头骨折的临床疗效。方法:自2009年3月至2012年2月对25例Mason II型,12例Mason III型桡骨小头骨折均采用切开复位BOLD钉内固定。结果:所有患者术后均获得随访12-24个月,平均随访14个月,肘关节功能评分:Mason II型平均分94分(88-98分),其中,优20例,良3例,可2例,差0例,优良率92%;Mason III型平均分91分(80-95分)其中,优9例,良1例,可2例,差0例,优良率83.3%。结论:BOLD钉治疗Mason II型及未累及桡骨颈的Mason III型桡骨小头有手术操作简单,固定稳定,允许早期活动等优点,可以作为这类骨折治疗的新选择。     

A method to average immunofluorescent histograms

Single parameter gated flow cytometric fluorescent histograms were obtained on normal donor blood mononuclear cells using several commonly available lymphocyte surface markers. A computer method was developed to average single parameter flow cytometric immunofluorescent histograms. The averaged histograms provide a means of pattern recognition for normal lymphocytes and will aid in the clinical evaluation of lymphocytosis. Averaged histograms may also serve as standards for more advanced analysis.     

Role of interleukin 1 and interleukin 2 in rat and mouse arthritis models

The production of interleukin 1 (IL 1) and interleukin 2 (IL 2) by macrophages and lymphocytes from three animal models commonly used for rheumatoid arthritis, viz. adjuvant-induced and type II collagen-induced rat arthritis, and MRL/1 murine arthritis was studied. Although the peritoneal macrophages from adjuvant-arthritic rats in culture produced increased amounts of prostaglandin E2 (PGE2) and lower levels of IL 1 than the control group, cells from collagen-arthritic rats released normal levels of PGE2, but increased amounts of IL 1. After activation with lipopolysaccharides, the IL 1 production by macrophages from all groups was comparable. Addition of indomethacin did not significantly change the IL 1 production in any of these groups. In the absence of any exogenous mitogen, IL 2 production by the lymphocytes of adjuvant-arthritic rats was low, but could be restored to the normal levels when phytohemagglutinin A (PHA) or concanavalin A (Con A) was added. The lymphocytes from collagen-arthritic rats were capable of producing IL 2 without the need of any T cell mitogen. The lymphocytes from MRL/1 mice seemed to lack the functionality in terms of IL 2 production. The macrophagic IL 1 production in these animals was normal. Our data suggest that the type II collagen arthritis model may closely resemble human rheumatoid arthritis in which IL 1 and IL 2 production by the mononuclear cells is significantly enhanced.     

The effects of cytocentrifugation on differential cell counts in samples obtained by bronchoalveolar lavage

Quantification of the differential cell count and total number of cells recovered from the lower respiratory tract by bronchoalveolar lavage is a valuable technique for the diagnostic study of interstitial lung diseases. To examine the effect on the cell counts of different methods of processing the lavage fluid, two comparisons were performed. First, two methods of differential cell counting were compared using 28 fluids. One count was performed in a Malassez hemocytometer after incubation of the living cells with neutral red for five minutes at room temperature; large cells and some small cells that had incorporated neutral red were identified as macrophages. Another count was performed on cytocentrifuge preparations made using the Shandon Cytospin I and Cytospin II and stained by the May-Grünwald-Giemsa method. The percentage of cells identified as lymphocytes was significantly lower on the cytocentrifuge preparations than with the Malassez hemocytometer. In the second study, the differential cell counts on smears prepared by the two types of cytocentrifuge (Cytospin I and Cytospin II) were compared for 32 bronchoalveolar lavage fluids. The percentage of small cells (especially lymphocytes) was lower on preparations made with the Cytospin I than on those made with the Cytospin II, but the difference was not significant. The results indicate that (1) cytocentrifugation of bronchoalveolar lavage fluids does result in a significant loss of small cells, especially lymphocytes, and (2) this loss is not significantly lessened by the use of the Cytospin II.     

The effect of a pattern in palmar interdigital II on a-b ridge count in black and white Down syndrome cases and controls

An unconfirmed study by Fang (Ph.D. thesis, Univ. of London, 1950) in Britain showed that individuals with Down syndrome had lower total a-b ridge counts in palmar Interdigital area II (ID II) than a group of controls. This study compares 603 white Down syndrome cases and 93 black Down syndrome cases with 668 white and 402 black controls. Our results confirm those of Fang in that the Down syndrome cases in both racial groups had lower total a-b ridge counts than their respective controls. In addition, the black controls and Down syndrome cases had lower a-b ridge counts than their white counterparts. The mean a-b ridge count was significantly lower in individuals with a pattern in ID II compared to individuals without a pattern in ID II in both the Down syndrome and control groups. Some of the lower a-b ridge counts in the Down syndrome samples can be accounted for by the fact that there is an increased frequency of a pattern in ID II in Down syndrome cases. Both Down syndrome and normal individuals who had a pattern unilaterally had a lower than expected a-b ridge count on the contralateral hand that did not have a pattern. There was a tendency also for increased asymmetry in Down syndrome cases with a pattern in ID II.     

Seasonal leukocyte and erythrocyte counts in fathead minnows

Leukocyte and erythrocyte counts on normal fathead minnows Pimephales promelas showed a marked seasonal trend. In a period from June to March, a three-fold increase in leukocytes was seen during August and September.     

Bronchoalveolar lavage in the assessment of pulmonary Hodgkin's disease

Abnormal chest radiographs in patients with Hodgkin's disease are occasionally due to pulmonary Hodgkin's disease. The fluids recovered from bronchoalveolar lavages (BALs) from 50 patients prior to autologous bone marrow transplantation for advanced Hodgkin's disease were examined. Abnormal chest roentgenograms were present in 24 patients (48%); 4 (17%) of these had Reed-Sternberg cells or their mononucleated variants in the lavage fluid and an alveolar lymphocytosis averaging 31.4% (normal: 11.5%). The lymphocytes were small and monotonous. Of the 20 patients with abnormal chest roentgenograms but no Reed-Sternberg cells in the lavage fluid, the lymphocyte count was 10.88%, with only 3 patients exceeding 17%. Two patients with normal chest roentgenograms had Reed-Sternberg-like cells in their lavage fluids and averaged 23% lymphocytes in their lavage differential count. Eosinophils averaged 1% or less of the lavage differential and were not predictive of pulmonary Hodgkin's disease. This experience suggests that pulmonary Hodgkin's disease can be diagnosed by BAL. Reed-Sternberg cells and their mononucleated variants can be recognized by their characteristic cytomorphologic features, although care must be taken not to misinterpret reactive binucleated macrophages as neoplastic cells. In patients with Hodgkin's disease, Reed-Sternberg cells should be sought when an alveolar lymphocytosis is present.     

Cell profiles in serial bronchoalveolar lavage after human heart-lung transplantation.

The aim of this study was to investigate serial changes in bronchoalveolar lavage (BAL) cell profiles after human heart-lung transplantation and to assess the clinical value of BAL cytology in the differential diagnosis of complications in the transplanted lung. BAL was performed serially on 23 occasions on four patients. Elevated counts of neutrophils (4-48%) were observed in all preparations, with peak values in the early postoperative phase, in bacterial infections and in cytomegalovirus pneumonitis. In the last condition, BAL cytology also showed relative lymphocytosis (less than or equal to 50%) with high proportions (less than or equal to 50%) of HLA DR-positive T lymphocytes. No characteristic light microscopic pattern was observed in acute pulmonary rejection. However, scanning electron microscopy revealed elevated counts (greater than 5%) of "villous" macrophages in BAL obtained during or shortly after episodes of rejection. BAL cytology may be helpful in differentiating viral and bacterial infections, while scanning electron microscopy seems to be more suitable to the diagnosis of acute pulmonary rejection.     

Suppression of antibody production by TNF-related apoptosis-inducing ligand (TRAIL)

TNF-related apoptosis-inducing ligand (TRAIL), a type II membrane protein belonging to the TNF family, induces apoptotic cell death in various types of tumor cells. However, little is known about its pathological and physiological functions in the immune system. In this study, we showed that administration of neutralizing anti-TRAIL mAb markedly increased serum auto-Ab levels, particularly of IgG1 subclass, in autoimmune-prone C3H/HeJ gld/gld mice without affecting lymphocytosis and lymphocytes populations. In an experimental system where TNP-specific Ab production was induced by immunization with TNP-modified syngeneic B lymphoma cells, expression of TRAIL on these cells significantly reduced TNP-specific Ab production, especially of IgG1 subclass, without affecting T cell priming. These results suggest a new role for TRAIL in the suppression of Ab production.     

Tolerance to class II major histocompatibility complex molecules is maintained in the presence of endogenous, interleukin-2-producing, tolerogen-specific T lymphocytes

We have examined the requirement for clonal reductions of tolerogen-reactive lymphocytes in mice of the A strain background rendered neonatally tolerant of class II major histocompatibility complex molecules. Tolerogen-specific mixed lymphocyte reactivity of lymphocytes obtained from 130 adult, class II tolerant mice, bearing a healthy skin allograft, was examined. Lymphocytes obtained from 86 mice responded to the tolerogen, in vitro, with a positive mixed lymphocyte response (MLR) indicating that a large proportion (75%) of adult class II tolerant mice on the A strain background are not clonally deleted for tolerogen-reactive lymphocytes. In addition, lymphocytes from 29 mice were MLR-negative to the tolerogen, and lymphocytes from 15 mice demonstrated such high amounts of proliferation to syngeneic stimulators that their specific response to the tolerogen could not be determined. In view of the discordance between the in vivo and in vitro expressions of tolerance in the MLR-positive mice, lymphocytes from these mice were compared with normal lymphocytes by several assays. 1) Tolerogen-specific proliferative responses obtained from both normal and tolerant lymphocytes could be inhibited by the addition of monoclonal antibodies specific for the relevant class II antigens; 2) quantitative differences in the ability of normal, as compared with tolerant cells, to respond to the tolerogen in the MLR were not apparent; 3) no evidence of qualitative differences in the cell-surface phenotype of the proliferating cell was observed, (i.e., the cells were Thy-1+, L3T4+, Lyt-2-); and 4) lymphocytes from both normal and MLR-positive tolerant mice produced substantial amounts of interleukin-2 in response to the tolerogen. Thus, clonal deletion of helper cells is not required for tolerance to class II major histocompatibility complex antigens and we propose that tolerance may be maintained by either 1) in vivo suppression of the tolerogen-specific helper cells or 2) selective deletion or suppression of class II specific effector cells.     

Pathogenesis of type II avian adenovirus infection in turkeys: in vivo immune cell tropism and tissue distribution of the virus.

Hemorrhagic enteritis virus (HEV), a type II avian adenovirus, causes intestinal hemorrhages and immunosuppression in turkeys. In this study, we exposed turkeys to virulent HEV and examined fractionated spleen cells for the presence of viral DNA by in situ hybridization and amplification of DNA extracted from virus-infected cells by PCR. HEV replication was detected only in the immunoglobulin M-bearing B lymphocytes and macrophage-like cells but not in the CD4+ or CD8+ T lymphocytes. The inability to infect T cells distinguishes type II avian adenoviruses from lymphotropic mammalian adenoviruses which infect and replicate in T cells. Furthermore, these data suggested that HEV-induced immunosuppression in turkeys may be due to the effect of the virus on B lymphocytes and macrophages. We also examined tissue tropism of HEV by in situ hybridization conducted on sections of lymphoid and nonlymphoid tissues. Large numbers of HEV-positive cells were detected in spleen and cecal tonsils. Diminutive viral activity was present in the intestines, the principal site of HEV-induced pathology. Thus, intestinal pathology was not associated with local cytopathic viral replication. This result and our previous observation that cyclosporin A abrogated intestinal hemorrhaging in HEV-infected turkeys strongly suggested that intestinal lesion induced by this virus may be immune system mediated.     

Glycosphingolipids of leukemic cells in adult T-cell leukemia-lymphoma

We analyzed lipids from leukemic cells of two patients with adult T-cell leukemia and compared them with those from T-cell lymphocytes of normal subjects. The neutral glycosphingolipids and gangliosides which were isolated were characterized by thin-layer chromatography and neuraminidase treatment. Both leukemic cells and normal lymphocytes had monoglycosylceramide and diglycosylceramide as major neutral glycosphingolipids. In one patient, diglycosylceramide was markedly increased. II3NeuAc-LacCer (GM3) and more complex gangliosides were detected in both cells. The most characteristic finding in leukemic cells was the occurrence of a disialylated ganglioside, II3(NeuAc)2-LacCer (GD3), which is not found in normal lymphocytes and neutrophils. This ganglioside may be due to the induced synthesis in association with malignant transformation.     

Cytologic diagnosis of peripheral T-cell lymphoma manifesting as ascites. A case report

BACKGROUND: Patients with malignant lymphoma seldom present with effusions without a known history of malignancy. Because of this, initial diagnosis of malignant lymphoma by effusion cytology is uncommon, with few reported cases. CASE: A 75-year-old male presented with fatigue, decreased appetite and progressively increasing abdominal girth over five weeks. Cytologic examination of ascitic fluid obtained by paracentesis revealed non-Hodgkin's lymphoma with a T-cell phenotype, confirmed by immunophenotypic and molecular studies. Approximately one week later, histologic examination of liver and bone marrow revealed involvement by lymphoma, demonstrating immunophenotypic and molecular profiles identical to those obtained from neoplastic lymphocytes recovered from the ascites fluid. CONCLUSION: This case demonstrates a rare presentation of peripheral T-cell lymphoma, clinically manifesting as ascites. In cases such as ours, where the effusion consists predominantly of small to intermediate-sized lymphocytes, distinguishing lymphoma from reactive lymphocytosis may be difficult. This case not only demonstrates the value of effusion cytology for lymphoma diagnosis but also illustrates how the use of various immunophenotypic and molecular techniques may assist the pathologist in properly diagnosing these difficult cases.