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Biomedical subjects

M L Labat

Publications and source records attributed to M L Labat.

At least 19 recordsLinked to original sources

Retroviruses and bone diseases.

In 1980, retroviruses were shown to be pathogenic to humans, and experimentation on animals involving retroviruses as causal agents of tumors and degenerative diseases of bone, brain, and lung gained interest. Osteopetrosis, which can be either inherited in rodents or retrovirally induced in cats, is exemplary. Because of replication cycle, retroviruses can be propagated not only as infectious agents but also as cellular genes. If a retroviral infection occurs in germ line cells, the viral genes, which must integrate in the host's DNA, can be passed on to the progeny and inherited as Mendelian characteristics. Therefore, a retroviral etiology could account for diseases that present either as sporadic (infectious) or familial (inherited), although they may be similar in their clinical manifestations. This approach led to the finding of 2 new human retroviruses: 1 in a patient who had sporadic benign osteopetrosis, and the other in a patient who had sporadic paraarticular osteoma. In both patients, the retrovirus was isolated from mononuclear blood cells, not from bone cells, because of the links between bone and the immune system. A systematic search for retroviruses in patients who have sporadic bone disease, which also may appear as inherited disease, has yet to be performed. Patients with sporadic disease could be managed by antiretroviral agents such as Zidovudin.

Animals

Bone marrow stromal cell defects and 1 alpha,25-dihydroxyvitamin D3 deficiency underlying human myeloid leukemias.

Primary myelodysplasia (MDP) and acute and chronic myelogenous leukemias (AML, CML) are considered disorders of clonal stem cell division. Several constitutive gene defects that contribute to the development of abnormal cell behavior have been identified in the hematopoietic cells. The role of bone marrow stroma cells in leukemogenesis, however, has not been established. We studied the organization of the bone marrow (BM) microenvironment to see if it was impaired during the initiation and progression of these malignancies. The buffy coat, hematon, and plasma fractions were separated from BM aspirates taken from healthy donors and diseased subjects at distinct clinical stages. The structural integrity of the BM microenvironment was evaluated analyzing the morphogenetic unit, the hematon. The hematon is a multicellular complex that includes fibroblasts, adipocytes, endothelial cells, resident macrophages, hematopoietic cobblestone area-forming cells (CAFC), high-proliferative potential colony-forming cells (HPP-CFC), granulocyte-macrophage colony-forming unit (GM-CFU), burst-forming unit erythroid (BFU-E), and terminally differentiated cells in normal BM. Hematon complexes were present in most BM aspirates from healthy donors (46H+/55). But they were absent from most of the patients with MDP (21H+/62) and AML (5H+/24) in the first perceptible phase, and from those with CML throughout the disease (5H+/55). Hematon complexes were present in the BM aspirate in 22/36 AML patients at clinical remission after chemotherapy or differentiation therapy. The hematon fraction isolated from normal BM, contained 25 times more 25-hydroxyvitamin D3 and about 500-fold more 1alpha,25-dihydroxyvitamin D3 than the BM plasma. The concentration of 1alpha,25-dihydroxyvitamin D3 was low or undetectable in the BM plasma of some, but not all, patients with MDP (18/35) or AML (9/24). Thus, in the BM microenvironment, the metabolism of low-density lipids and lipophylic hormones are severely impaired prior to initiation or during the accelerated expansion of leukemia cells. The lack of organized stromal network and the decreased level of some lipophylic hormones, acting probably as morphogens, may contribute to the onset and progression of human myeloid leukemias.

Bone Marrow

Retroviral expression in a patient who has paraarticular ossification.

Reverse transcriptase activity is reported in the mononuclear blood cells isolated from a patient in whom paraarticular ossification developed after surgery for an aneurysmal bone cyst. The enzyme was purified to apparent homogeneity by chromatography before being characterized biochemically for its template specificity and ionic requirement. The enzyme was able to transcribe poly(rA).(dT)12-18 very efficiently in the presence of Mn++ ions. Viral particles were observed in the HuT-78 cell line, cocultured with the mononuclear cells of the patient. No viral particles were observed in HuT-78 cells before the coculture. The patient was found seronegative for HIV-1, HIV-2, and HTLV-1. These results suggest that a new retrovirus infecting mononuclear blood cells may be involved in the development of ectopic ossification. This hypothesis is strengthened by the previous finding of a retrovirus in the mononuclear blood cells of a patient with benign osteopetrosis, and by the fact that HTLV-1 infected T-lymphocytes acquire the ability to secrete factors responsible for the lytic bone lesions observed in the patients. A family of human bone diseases that reflect T-cell dysfunction(s) and are caused by lymphotropic viruses may exist.

Bone Cysts, Aneurysmal

Monocytic origin of fibrosis. In vitro transformation of HLA-DR monocytes into neo-fibroblasts: inhibitory effect of all-trans retinoic acid on this process.

We report here the spontaneous in vitro transformation of blood monocytes into fibroblasts in a patient who developed pulmonary fibrosis following ciclosporin-mediated immunosuppression, necessitated by heart transplantation. The blood monocytes with this capacity expressed HLA-DR specificity. Monocytes/macrophages were identified by immunofluorescence using monoclonal antibodies against a specific monocyte/macrophage antigen, while the neo-fibroblasts were identified by electron microscopy and immunofluorescence using monoclonal antibodies against a cytoplasmic enzyme specifically involved in the synthesis of collagen. The secretion of collagen was demonstrated using antibodies against collagen. Both the monocytes/macrophages and the neo-fibroblasts express macrophage and fibroblast markers and are able to synthesize collagen. The all-trans retinoic acid derivative (all-trans RA) inhibits this in vitro transformation of HLA-DR monocytes/macrophages into neo-fibroblasts. Therefore, the possible therapeutic role of all-trans RA in controlling the development of fibrosis remains open to investigation. Until now, no efficient therapy is known for fibrotic diseases which are often lethal when affecting the lungs.

Aged

Immune cell defects affect bone remodelling in osteopetrotic op/op mice.

The aim of the present work was to further characterize immunological defects in osteopetrosis. The op/op mutant mouse is of particular interest since a marrow cavity develops spontaneously in older animals. The interleukin production (IL-1, IL-2, IL-3, IL-4, IL-6), the presence of macrophage differentiation antigens and the evolution of the bone lesions were studied in osteopetrotic and normal mice. Low levels of IL-1, IL-3 and IL-4 were observed at the age of 6 weeks in the op/op mice. However, at 22 weeks of age, the level of IL-1 and IL-4 returned to normal value in these op/op mice whereas the level of IL-3 remained partially decreased at the same age. Furthermore, macrophage expression of MAC-2 antigen, reduced at 12 weeks of age was found to be normal 10 weeks later. These immunological defects and their recovery seems to be concomitant with the healing of the bone lesions.

Animals

T-lymphocyte control of HLA-DR blood monocyte differentiation into neo-fibroblasts. Further evidence of pluripotential secreting functions of HLA-DR monocytes, involving not only collagen but also uromodulin, amyloid-beta peptide, alpha-fetoprotein and carcinoembryonic antigen.

Previous studies led us to demonstrate in pathological situations that the fibroblast, not the macrophage, was the terminal maturation step of the HLA-DR monocyte and that the entire process came under T-lymphocyte control. Fibrosis which developed under immunosuppressive treatment (cyclosporin) after organ transplantation is an illustration of these in vitro observations. The present in vitro study was undertaken in order to investigate whether or not this transformation process takes place under physiological conditions and if so, the nature of the T-lymphocyte control. We report that normal HLA-DR monocytes/macrophages are able to secrete type 1 collagen and to differentiate into neo-fibroblasts. However, contrarily to what happened in pathology, only a few neo-fibroblasts developed transiently. The addition of conditioned medium (CM) from activated T-lymphocytes greatly enhanced the transformation process. Counteracting this CM effect, cell-to-cell contact between neo-fibroblasts and T-cells resulted in the loss of fibroblastic shape. The 'end-result' macrophage engulfed numerous lymphocytes giving rise to a multinucleated cell. This giant cell no longer adhered to the slide and died. The question is raised as to whether the process observed in vitro is involved in vivo in tissue repair. We also report that HLA-DR monocytes and the neo-fibroblasts which derive from them are able to secrete, in addition to type 1 collagen, a variety of proteins such as uromodulin, amyloid-beta peptide, alpha-fetoprotein and carcinoembryonic antigen. In cystic fibrosis we previously reported a high level of uromodulin production by HLA-DR monocytes differentiating towards the fibroblastic phenotype. Pathologies characterized by excessive production of either alpha-feto-protein, carcinoembryonic antigen, beta-amyloid protein (Alzheimer's disease) should be investigated, taking into account the involvement of HLA-DR monocytes and their possible uncontrolled differentiation into neo-fibroblasts.

Amyloid beta-Peptides

Combined differentiation therapy in myelodysplastic syndrome with retinoid acid, 1 alpha,25 dihydroxyvitamin D3, and prednisone.

The myelodysplastic syndrome (MDPS) provides an opportunity for identifying host factors (genetic, endocrine, immune) involved in initiation and progression of preleukemia into frank acute myeloid leukemia. The aim of this study was to identify bone marrow (BM) cellular and humoral dysfunctions central to the development of MDPS and useful in therapeutic follow-up studies. Our preclinical studies have shown that (1) the characteristic stromal cell composition of the normal BM microenvironment was impaired in MDPS and in AML in 67 and 86% of the cases, respectively; (2) the 1 alpha,25(OH)2D3 concentration in BM plasma was abnormal in 50% of MDPS and 30% of AML; and (3) an inverse correlation existed in MDPS between the 1 alpha,25(OH)2D3 concentration and the frequency of F-CFU, (r = 0.41, p < 0.02), suggestive of a regulatory interaction between this secosteroid hormone and BM stromal cells. The analysis of clonal extinction of BM blast cells in response to all trans retinoic acid (RA), 1 alpha,25(OH)2D3, and colony stimulating factors (PHA-LCM), either alone or in various combinations, revealed individual patterns of responses in the cases of MDPS or AML. The results indicate the necessity for preclinical studies to select patients for combined differentiation therapy. Our ongoing clinical trials suggest that RA (Roaccutan, 20 mg/day continuously) as induction therapy, followed at weeks 6 to 8 by prednisone (40 mg/day for 15 days) and 1 alpha,25(OH)2D3 (Rocaltrol, 3 x 0.25 micrograms/day for 3 months) may induce a long-lasting hematological remission in MDPS.

Bone Marrow

Cystic fibrosis: production of high levels of uromodulin-like protein by HLA-DR blood monocytes differentiating towards a fibroblastic phenotype.

We report here the spontaneous in vitro transformation of blood monocytes into fibroblasts, in a patient suffering from cystic fibrosis (CF). The blood monocytes with this capacity express HLA-DR specificity. Monocytes were identified by non-specific esterase activity and by immunofluorescence using monoclonal antibodies against monocytes/macrophages antigens. Neo-fibroblasts were identified by electron microscopy and immunofluorescence using monoclonal antibodies against a cytoplasmic enzyme specifically involved in the synthesis of collagen. The secretion of collagen was evidenced using antibodies against type I collagen. Both monocytes/macrophages and neo-fibroblasts express the monocytic and the fibroblastic markers and synthesize type I collagen. This transformation observed in vitro might mimick the process of fibrosis development which takes place in vivo, particularly in pancreatic acini, lungs and intestine of patients with CF. Interestingly, the whole process in vitro is inhibited when T lymphocytes are properly stimulated by IL2. In addition, both monocytes and neo-fibroblasts secrete high quantities of uromodulin-like glycoprotein. The significance of this finding is discussed in relation to the thick mucus secretion which characterizes the disease. In addition, from a fundamental point of view, it confirmed in a large series of patients that this observation may have significant implications, since CF mutation impairs the gene coding for cAMP-regulated Cl- channel and that it has been proposed that uromodulin might be implicated in Cl- transport. Therefore the question of the relationships between uromodulin and the cAMP-regulated Cl- channel arises.

Adolescent

Monocytic origin of fibroblasts: spontaneous transformation of blood monocytes into neo-fibroblastic structures in osteomyelosclerosis and Engelmann's disease.

We describe here two pathological situations, osteomyelosclerosis and Engelmann's disease, in which HLA-DR blood monocytes modulate to the fibroblastic class, in long-term culture. Monocytes/macrophages were identified by immunofluorescence, using monoclonal antibodies against surface markers (Leu M3, CD 68, and HLA-DR) and the neo-fibroblasts by electron microscopy and immunofluorescence using monoclonal antibodies against a cytoplasmic enzyme specifically involved in the synthesis of collagen (5B5). Macrophages makers were found on the neo-fibroblasts, whereas HLA-DR macrophages expressed the cytoplasmic marker 5B5. Since osteoblasts are classically derived from fibroblasts, the significance of the in vitro differentiation of monocytes/macrophages into fibroblasts to the in vivo mechanism leading to excessive osteoblastic proliferation in both osteomyelosclerosis and Engelmann's disease, is discussed. The possible involvement of this pathway leading from monocytes to fibroblasts and osteoblasts in the normal process of bone modeling and remodeling in questioned.

Camurati-Engelmann Syndrome

Retinoic acid in mono- or combined differentiation therapy of myelodysplasia and acute promyelocytic leukemia.

Myelodysplastic preleukemic syndromes (MDPS) and acute promyelocytic leukemia (APL) share a surprising in vivo sensitivity to the hormonally acting 13 cis or all trans retinoic acids (transRA). Here we show that transRA as a monotherapeutic agent induced a stable remission in APL at the third relapse. In MDPS, treatment with prednisone and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25D3) 13 cis RA induced a long-lasting hematological remission. Initially both patients had an impaired BM microenvironment which regenerated on retinoid therapy as judged by reappearance of the Hematon fraction in the BM aspirates. Our preclinical experiments using long-term liquid BM cultures (LTBMC) indicated that several individual patterns of growth and differentiation responses can be induced by combinations of transRA, 1 alpha,25D3 and hemopoietic growth factors (HGFs). The biological responses may vary from complete clonal extinction to a significant growth stimulation of the leukemic blast cell populations. These results further support the importance of preclinical studies in selecting "good" responders for, and excluding "poor" responders from protocols using differentiation therapy.

Bone Marrow

A new approach to the study of the origin of genetic diseases: retroviral etiology of osteopetrosis.

The recent evidence of retroviral activity in a patient suffering from an atypical form of benign osteopetrosis suggests that it may represent the ancestral infectious form of a disease recognized until now only as a genetic disease. Therefore, making c-DNA probes from that virus should evidence retroviral-like sequences integrated in the genome of patients suffering from the inherited form of the disease. One of the goals of these investigations is to use these probes to map human "osteopetrosis" mutation(s) in the genome. A new approach is therefore defined for the study of these diseases, which although similar in their clinical manifestations, appear either as "sporadic" or "familial" cases: a retroviral etiology can account for this discrepancy. Another approach is also suggested for these kinds of disease, consisting of the search for retroviral sequences in a gene coding for an enzyme, when the main disease is associated with an apparently unrelated enzymatic deficiency. The insertion of retroviral sequence in a gene coding for an enzyme may result in both the disease and the inactivation of the gene. By comparing the genomic sequence of the enzyme in normal and pathological cases, the integrated retroviral gene(s) sequence will be available.

DNA Probes

Retroviral expression in mononuclear blood cells isolated from a patient with osteopetrosis (Albers-Schönberg disease).

We report the presence of reverse transcriptase activity in the supernatant of long-term culture of mononuclear blood cells (monocytes and lymphocytes) isolated from a 27-year-old patient suffering from benign osteopetrosis. The enzyme was purified to homogeneity according to the technique of Chandra and Steel, by chromatography, first on DEAE-cellulose (DE 52) and then on phosphocellulose (P11). After purification, the enzyme was characterized biochemically for its template specificity and ionic requirements. The purified enzyme was able to transcribe poly(rA).(dT)12-18 and poly(rC).(dG)12-18 very efficiently and had a marked preference for Mg2+ ions over Mn2+ ions. The pattern of ionic dependency for this enzyme is similar to that of reverse transcriptases purified from human lymphotropic viruses. The patient was tested and found sero-negative for HIV-1, HIV-2, and HTLV-I and seropositive (immunoglobulin G) for cytomegalovirus. Epstein-Barr virus nuclear antigens (EBNA) were detected in the patient's B lymphocytes. Since reverse transcriptase is the hallmark of retroviruses, we suggest that a retrovirus may be involved in the etiology of osteopetrosis.

Adult

Retroviruses, immunosuppression and osteopetrosis.

Since decades, retroviruses are known to induce the formation of dense bones (osteopetrosis) in animals, either by increasing osteoblastic proliferation (ALVs viruses) or by decreasing osteoclastic bone resorption (FeLV virus). The latter is a good model of the inherited disease since neonatally infected cats die from wasting disease, like mutant op/op rats and children suffering from juvenile-malignant osteopetrosis. These similarities have prompted this review of retrovirus-induced osteopetrosis in animals. We suggest the possibility that retroviruses might be involved, at least in part, in the induction of the human disease.

Animals

Excess of lympho-reticular cell complexes in the bone marrow linked to T cell mediated dysmyelopoiesis.

The term dysmyelopoietic syndrome (DMPS) covers a variety of closely related disorders with various etiological factors, and characterized by chronic (pan) cytopenias whose prognosis and treatment are still controversial. Despite the recent efforts to identify pathogens, effector cells and soluble cell products involved in the development of this syndrome only a few comprehensive experimental data are available, useful for elaboration of therapeutic regimens. We describe here a patient with DMPS who was refractory to inductive chemotherapy. Initial agar gel culture studies revealed the activity of hematopoiesis inhibitory T cells within the bone marrow that could be suppressed by prednisone both in vitro and in vivo. However another pathological cell features, the excess of an unusual lympho-reticular cell complexes was identified in long-term liquid cultures. These symbiotic cell complexes persisted throughout the disease, despite the prednisone induced hematological remission, suggesting their causative role in the disease, that more recently progressed towards acute myeloid leukemia.

Adult

[Osteopetrosis].

Animal models of osteopetrosis have led us to consider this condition to be an immune disease. In both the hereditary from in the op/op mutant rat and in experimentally induced from by Cl2MDP, osteopetrosis is associated with immune deficiencies, such as the inhibition of the mitogenic response of T lymphocytes, the inability to develop adjuvant arthritis. In particular, the differentiation of the thymus dependent macrophages is inhibited in the mouse treated with Cl2MDP. These animal models illustrate the relationships between the immune system and the bone regeneration system. They enable the study of the relationship between T lymphocytes, macrophages and osteoclasts.

Animals

Dichloromethylene diphosphonate (Cl2MDP) reduces natural killer (NK) cell activity in mice.

Daily administration of dichloromethylene diphosphonate (Cl2MDP) to (C57BL/6 X DBA/2) F1 hybrid mice, from two days of age (10 mg of P/kg body weight), resulted in a marked impairment of natural killer (NK) activity of spleen cells against YAC-1 lymphoma cells. The suppressive effect increased with the duration of the treatment. Cessation of the treatment led to a rapid recovery (in 2 weeks) of NK activity while the osteopetrotic bone lesions persisted. Thus, the loss of natural killing cannot be explained by the simple reduction of bone marrow volume secondary to Cl2MDP-induced osteopetrosis. However, as NK cells are considered to be dependent on the bone marrow because they cannot be sustained by extramedullary production, a direct effect of Cl2MDP on the generation of NK cell precursors by the bone marrow was not excluded. Cl2MDP was not directly toxic to the fully differentiated splenic NK cells, since the addition of Cl2MDP to the in vitro assay (10(-5)-10 micrograms/ml) did not reduce cytotoxicity. These studies suggest that impairment of NK activity during Cl2MDP treatment may have clinical toxicologic implications since NK cells have been suggested to play an important role in natural host defenses against infection and neoplasia.

Animals

(Dichloromethylene)diphosphonate-induced impairment of T-lymphocyte function.

The daily subcutaneous administration of (dichloromethylene)diphosphonate (clodronate) to 3-day-old normal inbred Wistar-Furth rats for 30 days produces osteopetrotic bone lesions resembling those of the osteopetrotic mutants. Furthermore, tooth eruption is prevented, growth slows down, and signs of runt disease appear. The weight of the thymus is decreased, and T cells from the thymus and spleen respond weakly to mitogens. These thymic disorders associated with defective bone resorption are very similar to those previously reported in the osteopetrotic mutant op rat and support the hypothesis of a link between the thymus and normal bone modeling and remodeling.

Alkaline Phosphatase

Lack of a T-cell dependent subpopulation of macrophages in (dichloromethylene) diphosphonate-treated mice.

In Cl2MDP-osteopetrotic mice, one subpopulation of thioglycollate-induced peritoneal exudate macrophages (M phi) is missing. This subpopulation is precisely the one whose differentiation is known to be dependent on T-lymphocytes, as it is also missing in the athymic nu/nu mice. Cl2MDP-induced osteopetrosis being partially attributable to deficient osteoclastic bone resorption, raises the possibility that this missing M phi subpopulation might represent the precursors of osteoclasts. It is suggested from this work that the interplays between T-cells and M phi, so well known in immunity and inflammation, may also be relevant to osteoclastic differentiation and therefore, to bone remodeling.

Animals