Cut-off values and significance of Oil Red O-positive cells in bronchoalveolar lavage fluid.

OBJECTIVE: To evaluate the percentage and predictive value of Oil Red O-positive macrophages (ORO-PM) to identify lipid-laden macrophages in bronchoalveolar lavage fluids (BALF) from patients with different pathologies. METHODS: The percentage and absolute numbers of ORO-PM were evaluated in 305 BALF. The patients were separated into ten groups: corticosteroid treatment (n = 18), amiodarone treatment (n = 8), interstitial fibrosis (n = 11), human immunodeficiency virus (HIV)-positive (n = 25), infectious pneumonia (n = 43), severe haematological disorder (n = 25), interstitial syndrome (n = 109), suspicion of cancer (n = 17), transplant recipients (n = 50) and controls (n = 43). The total and differential cell counts in BALF were recorded. The presence of specific pathogens was also noted. Parametric and non-parametric tests were used to compare the values between groups. Receiver-operating characteristics (ROC) curves were established in order to determine a cut-off value. RESULTS: The percentages of ORO-PM were (mean +/- standard deviation) 21.67 +/- 29.12 in the corticosteroid group, 10.00 +/- 12.49 in the amiodarone group, 19.45 +/- 20.72 in the interstitial fibrosis group, 47.80 +/- 30.46 in the HIV group, 19.72 +/- 26.26 in the infectious pneumonia group, 27.42 +/- 30.04 in the severe haematological disorder group, 25.18 +/- 30.63 in the interstitial syndrome group, 17.64 +/- 27.76 in the suspicion of cancer group, 22.50 +/- 27.27 in the transplanted recipients group and 2.63 +/- 3.48 in the control group. Significantly higher values were found in all groups when compared with the control group (P < 0.001). Only the HIV group showed higher numbers of ORO-PM when compared with the interstitial syndrome group (P < 0.01). According to ROC curves, > 6% ORO-PM was suggested as the positive cut-off value. CONCLUSION: Significantly increased numbers of ORO-PM were associated with various lung pathologies. However, the higher numbers observed in HIV patients require further investigations.

A new method for encapsulation of living cells: preliminary results with PC12 cell line.

A new method is described for encapsulation of living cells. PC12 rat adrenal pheochromocytoma cells, which have been shown to synthesize, store and release dopamine were employed. The particles are made first and the cells then incorporated in a gentle mechanical procedure. The morphology (by light and electron microscopic observation), stability, rheology, texture and permeability of these microcapsules provided by Kappa Biotech were investigated. Membrane permeability studies demonstrated exclusion of 69,000 Da human serum albumin, but equilibrium of D-glucose and inulin was within 24h, indicating a molecular weight cut-off in the 5000-70,000 Da range. The viability and the function of the encapsulated cells were evaluated by measuring the spontaneous release of dopamine by high performance liquid chromatography with electrochemical detection. The results show that dopamine-secreting cells can be sequestered in a semi-permeable capsule and still display good viability and proliferation for at least 1 month.

[Management of intractable cancer pain: from intrathecal morphine to cell allograft]. / Traitement des douleurs chroniques rebelles d'origine cancéreuse: de la morphinothérapie intrathécale à la thérapie cellulaire.

The durable effectiveness of intrathecal morphine administration is well established for the management of intractable cancer pain, after failure of systemic opioids, secondary to the persistence of non-reversible undesirable side effects. Many patients are referred to late in the disease course. This conservative method to control pain of malignant origin must not be reserved for last resort treatment for terminal patients. Intra-cerebro-ventricular morphine administration is a very effective and generally safe method for controlling intractable cancer pain. Because of the chronic implantation of an intra-ventricular catheter this method is somewhat invasive. Its indications remain a simple and effective alternative when the topography of nociceptive pain is diffuse or cephalic. In clinical practice, intrathecal and/or intra-cerebro-ventricular administration of opioids is limited by cost, the need for specialized maintenance and mechanical malfunctions if implantable drug delivery systems, or by the risk of bacterial contamination and ambulatory constraints when repeated daily injections via an intrathecal access port are used. To answer these limitations, cell therapy using intrathecal chromaffin cell allograft is a promising approach for the management of cancer pain refractory to traditional drug therapy and pain lesion surgery. The basic rationale and preclinical studies on experimental pain models have enabled starting prospective clinical trials. Prior to transplantation, handling and preparation of the chromaffin tissue is critical for allograft viability. The initial results of clinical trials with human chromaffin cell grafts from intractable cancer pain have reported long-lasting pain relief, in correlation with met-enkephalin release into the CSF. Convincing evidence will require controlled studies. The limitations of this innovative cell therapy and especially the lack of human adrenal gland availability point to the need for new sources of cells. Perspectives include xenogenic or engineered cell lines.

Adrenal medullary explants as an efficient tool for pain control: adhesive biomolecular components are involved in graft function ex vivo.

Adrenal medullary (AM) tissue transplantation into the central nervous system has been reported as a potential source of opioid peptides and catecholamines, which have analgesic effects useful in the control of chronic pain. Clinical trials, involving allogeneic graft of whole tissue explants into the subarachnoid space of the lumbar spinal cord, have already been reported. The aim of the present study was to determine whether adhesion and function of AM explants were related in some extent and how this relationship could account for improvement of AM tissue in terms of analgesic activity before grafting. Our experiments demonstrated a significant correlation between the adherent state of AM organoids during culture and a sustained secretion of Met-enkephalin and catecholamines by chromaffin cells (CC). These findings suggest that optimal culture condition for AM organoid adhesion can be defined for maintenance of tissue, prior to transplantation. Using immunocytochemistry, flow cytometry, and ELISA assays we showed that different cell adhesion molecules (CAMs) and extracellular matrix ECM proteins were expressed and released by AM cells during culture. Adherent AM organoids expressed increased levels of specific neural CAMs (NCAM and HNK-1 epitope) and integrin chains (beta1, alpha1, alpha2, alpha4, alpha5) and deposited markedly higher levels of fibronectin, but also laminin and collagen IV. Those molecules and probably adhesion processes they control might be involved in the maintenance of the CC-secreting neuroendocrine phenotype through cellular signaling pathways.

[Clinical perspectives of xenografts: encapsulated chromaffin cells and pain]. / Perspectives cliniques des x¿enogreffes: cellules chromaffines encapsul¿ees et douleur.

Intrathecal allograft of chromaffin cells can be effectively used in replacement of more conventional therapies for treating intractable chronic pain, such as in cancer. The efficacy of this technique depends on the ability of those cells to produce analgesic opioids and on the immuno-privileged property of the central nervous system, in which rejection risks are limited. However, there are some limitations to the generalization of this new therapy, mainly due to the low number of available grafts. Thus other sources than humans have to be considered. Here we discuss the pros and cons of the xenogeneic chromaffin cells of bovine or porcine origin. Graft immuno-isolation, for example, by using cell encapsulation, seems to be unavoidable in spite of the graft site.

Intrathecal grafting of unencapsulated adrenal medullary tissue can bring CD4 T lymphocytes into CSF: a potentially deleterious event for the graft.

Adrenal medullary tissue including chromaffin cells was grafted intrathecally in cancer patients to relieve intractable pain. The central nervous system (CNS) is considered an immune privileged site. Therefore, non-HLA-matched and unencapsulated tissue was grafted in 15 patients and 1 sham control in a series of at least 20 grafts. We observed an increase in CSF lymphocyte counts in 15/20 allografts (75%). In contrast to peripheral blood, CD4 T cells predominated in the CSF, but failed to exhibit an activated phenotype (CD25+ CD45RO+ HLA-DR+). The positive effect of graft on pain, the high met-enkephalin levels, the absence of any increase in CSF cytokine levels particularly for IFN-gamma or IL-2 (but not IL-10 and IL-6), indirectly indicated that the graft was tolerated despite the presence of CSF lymphocytes. The single treatment failure and three of four cases of partial efficacy occurred in grafts where CSF lymphocytes were present. Moreover, when assayed (n = 7), the CD4+ CSF lymphocytes still retained the capacity to exhibit ex vivo a normal or enhanced frequency of T CD4 cells producing IFN-gamma and IL-2. Taken together, our observations indicate that impairment of the local immunosuppressive balance can lead to activation of those CSF CD4 T cells and drive a rejection process. This study suggests further work on the purification and/or the immunoisolation of tissues grafted in the CNS will be necessary, particularly when the possibility of long-term and repeated grafting is considered.

One-year chromaffin cell allograft survival in cancer patients with chronic pain: morphological and functional evidence.

The control of chronic pain through transplantation of chromaffin cells has been reported over the past few years. Analgesic effects are principally due to the production of opioid peptides and catecholamines by chromaffin cells. Clinical trials have been reported with allografts consisting of whole-tissue fragments implanted into the subarachnoid space of the lumbar spinal cord (14,19,36). In the present study, allogeneic grafts were successfully used to control chronic pain in two patients over a period of 1 yr based on patient reported pain scores, morphine intake, and CSF levels of Met-enkephalin. Macroscopic examination at autopsy located the transplanted tissue fragments in the form of multilobulated nodules at the level of the spinal axis and cauda equina. Immunocytochemical microscopy showed neuroendocrine cells are positive for chromagranin A (CGA), and enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH). The results suggest that there is a relationship between analgesic effect, Met-enkephalin levels in CSF, and the presence of chromaffin cells surviving in spinal subarachnoid space.

Relief of intractable cancer pain by human chromaffin cell transplants: experience at two medical centers.

In addition to its possible role as a replacement source in CNS degenerative diseases, neural transplantation may be used to augment the normal production of neuroactive substances. Our laboratory at the University of Illinois at Chicago has shown, in both acute and chronic pain models, that transplantation of adrenal medullary tissue or isolated chromaffin cells into CNS pain modulatory regions can reduce pain sensitivity in rodents. Chromaffin cells were chosen as the donor source since they produce high levels of both opioid peptides and catecholamines, substances which reduce pain sensitivity when injected locally into the spinal subarachnoid space. The analgesia produced by these transplants probably results from the release of both opioid peptides and catecholamines since it can be blocked or attenuated by both opiate and adrenergic antagonists. Studies indicate that even over long periods there is no apparent development of tolerance. Promising results have been obtained in preliminary clinical studies using allografts of adrenal medulla to relieve cancer pain. This clinical review encompasses results at two Medical Centers-University of Illinois at Chicago and University Paul Sabatier, Toulouse, France-in assessing efficacy of subarachnoid adrenal medullary transplantation for alleviating cancer pain. Our clinical and autopsy data strongly support our previous laboratory studies, i.e., that chromaffin cell transplants into the subarachnoid space represent a promising new approach to the alleviation of chronic pain. It is suggested that further clinical studies are now warranted.

Transplantation of human chromaffin cells for control of intractable cancer pain.

Adrenal medullary chromaffin cells produce high levels of endogenous opioid peptides. Recent data suggest that transplantation injected locally into the spinal subarachnoid space reduced intractable malignant pain. In order to determine the feasibility, the efficacy and the risks of using adrenal medullary tissue for control of irreducible pain, we have developed a transplantation protocol on cancer pain patients selected when they required chronic intrathecal injection of morphine and progressively increasing doses to maintain the level of analgesic effects. At the present time, our clinical trial involves 8 patients. We report here our initial results (mean follow-up: 5 months). The various data collected before and after the intrathecal administration of chromaffin cells included: 1) Pain evaluation over time, with concomitant narcotic intake, 2) CSF sampling through an implanted access port to determine the following biological parameters: biochemical assay for opioid peptides, cell count and phenotyping of lymphocytes, 3) peripheral blood samples for lymphocyte typing. The results confirm the efficacy of adrenal medullary transplantation into spinal CSF for controlling irreducible cancer pain. Complementary intrathecal and oral morphine were totally stopped in 2 cases and stabilized in 5 others. It seems essential to have an important volume of grafted tissue to achieve analgesia with high levels of metenkephalin in CSF. A progressive decrease in metenkephalin release was observed from 2 to 4 months after the transplantation. Two patients with a long-term follow-up (8 and 12 months) needed another intrathecal chromaffin cell graft.

Influence of a long duration exposure, 69 months, to the space flight factors in Artemia cysts, tobacco and rice seeds.

Three french laboratories have participated in the Free Flyer Biostack experiment. Artemia cysts, tobacco seeds and rice caryopsis and embryos were used. Biological objects in monolayers were dead. In opposite, a large fraction of samples used in bulk survived. A stimulatory effect occurred in the first steps of development in Artemia cysts. In fact, the larval survival was unchanged or slightly reduced. In tobacco a drastic decrease in germination and survival rate was observed. Space flight did not induce genetic changes. In rice, results depend on the variety which was investigated; the growth rate stimulation in flight samples is discussed with respect to controls.

Antibodies to bladder-tumor-associated antigens as prognosis probes in the flow-cytometric analysis.

Monoclonal antibodies directed against bladder tumor cells (10D1, 7C12, 6D1, 3C6, G4 and E7) and human leukocyte antigen (HLe1) were tested by flow cytometry on 68 bladder tumors involving 10 grade I, 29 grade II and 29 grade III tumors (WHO classification). According to their evolution stage, these tumors can be subdivided into 17 stage Pa, 34 stage P1, 7 stage P2 and 10 stage P3. Fifteen normal bladder samples were used as a control. Analysis of DNA content revealed a first group of 31 tumors with a unimodal DNA profile. In the second groups of 37 tumors, the DNA profile was bimodal. Cells from grade I tumors were labelled with 10D1 and 6D1 antibodies; all these cells showed a unimodal DNA profile. Grade III tumor cells were labelled with antibodies G4 and E7; most of these cells showed a bimodal DNA profile. The percentage of HLe1-positive cells decreased with the pathological grade and stage of tumor. The composition of infiltrating leukocytes was different in unimodal and bimodal tumors. In conclusion, cells of low-grade tumors can be identified with 10D1 and 6D1 antibodies, and antigens recognized by G4 and E7 antibodies are mostly expressed by aneuploid cells. HLe1 antibody demonstrates the importance of the inflammatory reaction in bladder tumors. Moreover, in flow cytometry, leukocytes within a tumor could be used as internal reference for precise measurement of the DNA content of tumor cells.

Degradation of yolk in the brine shrimp Artemia. Biochemical and morphological studies on the involvement of the lysosomal system.

The degradation of yolk granules during the development of Artemia was studied. The results obtained suggest that lysosomes are involved in the process. In homogenates of embryos and larvae at different stages of development, the distribution of 2 lysosomal markers, acid phosphatase and cathepsin B, was studied by sucrose isopycnic gradient centrifugation. Three peaks of enzyme activity of densities greater than 1.3 and around 1.25 and 1.18 were observed. As revealed by electron microscope analysis, the 3 peaks were found to be associated with increasingly degraded yolk structures which stained for acid phosphatase. The process can be mimicked in vitro by incubating isolated yolk granules and lysosomes. The enzyme activity levels of the 3 peaks observed during development presented an oscillatory pattern, suggesting that degradation of yolk is cyclic. Five cycles of degradation were observed during the initial 60 hr of development.

Lymphoid cell lines as a model system for the study of Wolman's disease: enzymatic, metabolic and ultrastructural investigations.

Epstein-Barr virus (EBV) transformed lymphoid cell lines (LCL) were established from blood lymphocytes of a patient affected with Wolman's disease (WD) and from her parents. These LCL showed a severe deficiency in acid lipase activity using every substrate in comparison to LCL from normal subjects, in which acid lipase activity was similar to that in blood lymphocytes. In the LCL from Wolman's disease a major accumulation of neutral lipids was observed, mainly cholesteryl esters, CE (amount around 7 times higher than in normal cells and ratio of esterified/free cholesterol increased by 10 times) and to a lesser extent triglycerides, TG (amount increased by 1.5 times). Electron microscopy showed the storage vacuoles of neutral lipids quite characteristic of this lysosomal disease. The reported data demonstrated the validity of transformed LCL as a cellular model system in culture for experimental studies of Wolman's disease and for investigating the lysosomal metabolism of neutral lipids.

Study of minimal inhibitory concentration of antibiotics on bacteria cultivated in vitro in space (Cytos 2 experiment).

The aim of the Cytos 2 experiment, carried out during the French-Soviet manned flight in July 1982, was to study the bacteria's sensitivity to antibiotics cultivated in vitro during the orbital flight, using the bacterial method of minimal inhibitory concentration (MIC). Two species of bacteria were tested with various antibiotics: Staphylococcus aureus with Oxacillin, Chloramphenicol and Erythromycin; Escherichia coli with Colistin and Kanamycin. The results show an increase in resistance to antibiotics particularly strong in E. coli and weaker in Staphylococcus aureus. Considering these results, we think that there might be a relationship between the increase in resistance to antibiotics and a stimulating effect on growth rate by the factors of environmental space.

New tools for the study of Niemann-Pick disease: analogues of natural substrate and Epstein-Barr virus-transformed lymphoid cell lines.

Acid sphingomyelinase activity was determined in Epstein-Barr virus-transformed lymphoid cell lines (LCL) established from patients affected with Niemann-Pick disease (NPD) using several substrates: sphingomyelin derivatives, radiolabeled [14C]sphingomyelin (SM), fluorescent N-(10-(1-pyrene)decanoyl)sphingomyelin (P10-SM) or colored trinitrophenylaminolauryl-sphingomyelin, and the chromogenic non-natural substrate 2-N-(hexadecanoyl)amino-4-nitrophenylphosphoryl-choline. LCL from NPD Type A and Type B showed a severe deficiency of acid sphingomyelinase determined using either substrate, whereas LCL from normal subjects had an activity close to that of blood leukocytes. Sphingomyelinase in normal LCL had the same pH optimum (5.0-5.2) and molecular form (pI 5.8) as the enzyme from other sources; identical profiles and activity levels were obtained using the various analogues of sphingomyelin. However, among these derivatives, the assay using P10-SM appeared as the most useful and sensitive for enzymatic diagnosis of NPD. Electron microscopy of NPD LCL demonstrated the lysosomal storage. These results prove the validity of LCL as an experimental model system for NPD.

Biochemical and ultrastructural findings in a lymphoid cell line from Niemann-Pick disease type A.

A lymphoid cell line (LCL) established by Epstein-Barr Virus (EBV)-transformation of blood B-lymphocytes from a patient affected with Niemann-Pick disease (NPD) Type A exhibited a severe deficiency of sphingomyelinase activity (less than 10% residual activity). Ultrastructural investigation showed in LCL from NPD type A, the presence of numerous osmiophilic, electron-dense inclusions with myelin-like figures characteristic of the accumulation of sphingomyelin (and other amphiphilic lipids) similar to those observed in tissues of patients affected with NPD.

Epstein-Barr virus transformed lymphoid cell lines as a new model system in culture for the study of GM2-gangliosidoses: Tay-Sachs and Sandhoff diseases.

Epstein-Barr Virus transformed cell lines (LCL) were established from blood B-lymphocytes of patients affected with GM2-gangliosidoses variant O (Sandhoff disease, SD) and variant B (Tay-Sachs disease, TSD). LCL from SD showed a severe deficiency of activity of the major lysosomal beta-N-acetylhexosaminidase isoenzymes, Hex A and B; the residual activity was due to Hex S and Hex C. In LCL from TSD, the whole Hex activity was not deficient but isoenzyme composition was completely abnormal. Ultrastructural investigations showed the presence of pleiomorphic enlarged lysosomes appearing as clear vacuoles containing a finely fibrillo-granular material characteristic of the visceral lysosomal storage of gangliosidoses.

Ultrastructural investigations on two lymphoid cell lines from Niemann-Pick disease type B.

Lymphoid cell lines (LCL) were established by Epstein-Barr Virus (EBV) transformation of blood B-lymphocytes from two different patients affected with Niemann-Pick disease (NPD) type B. Those lines were severely deficient in sphingomyelinase activity (8% and 10% residual activity). Ultrastructural investigations showed in both these lines the presence of numerous osmiophilic, dense and pleiomorphic inclusions characteristic of lysosomal storage (due to the accumulation of amphiphilic lipids) similar to those observed in tissues from NPD.

[Biological effects of weightlessness at the cellular level. Comparative study of cultures of Paramecia aboard the orbital station Salyut-6 and a stratospheric balloon]. / Effets biologiques de l'impesanteur à l'échelon cellulaire. Etude comparative de cultures de paramécies à bord de la station orbitale Saliout-6 et d'un ballon stratosphérique.

In order to distinguish the effects of cosmic rays from those of weightlessness at the cellular level, we performed experiments aboard stratospheric balloon, where gravity is equal to 1 g and cosmic radiation roughly equal to that aboard Salyut-6. The results suggest that the stimulation of cell proliferation is probably due to cosmic rays, metabolic changes being related to microgravity.