A promoterless period gene mediates behavioral rhythmicity and cyclical per expression in a restricted subset of the Drosophila nervous system.

Transgenic flies carrying a 7.2 kb piece of DNA from the period (per) gene were analyzed for the presence of circadian locomotor activity rhythms and fluctuations of per-encoded mRNA and protein. The 5' end of this genomic fragment is within the first intron, which precedes the coding region. This promotorless fragment could rescue circadian behavioral rhythms and mediate spatial expression of PER in a subset of wild-type per cells within the CNS and PNS. In one behaviorally rhythmic line, PER protein was found in only "per lateral neurons." In the rhythmic transgenics, per mRNA and protein levels undergo circadian cycling, as previously described for wild type. Cycling of PER in brain cells of flies carrying the same 7.2 kb piece of per DNA under the control of a heat shock promoter corroborated the hypothesis that per's molecular cyclings and behavioral rhythmicity are causally related.

Layersome: development and optimization of stable liposomes as drug delivery system.

This paper describes the development of stable drug delivery systems named layersomes. The layersomes are conventional liposomes coated with one or multiple layers of biocompatible polyelectrolytes in order to stabilise their structure. The formulation strategy is based on an alternative coating procedure of positive poly(lysine) (pLL) and negative poly(glutamic acid) (pGA) polypeptides on initially charged small unilamellar liposomes (SUVs). The size distribution and the zeta potential of the final entity depend on the number of polyelectrolyte layers and the charge of the last coating layer. Morphological studies were achieved by flux cytometry and cryo electron microscopy. Release studies of encapsulated hydrophilic 5(6)-carboxyfluorescein (5,6CF) in the presence of Triton or ethanol showed an increased membrane resistance of the layersomes compared to classical SUVs. Finally, encapsulation of piroxicam (PX) was performed with success.

Step-by-step build-up of covalent poly(ethylene oxide) nanogel films.

Hydrogels based on poly(ethylene glycol) (PEG) are commonly used for studies related to cell fate and tissue engineering. Here we present a new covalent layer-by-layer build-up process leading to PEG coatings of nanometer size called "nanogel films". Compared to macroscopic hydrogels, such nanogels should provide a fine control over the structure and the thickness of the coating. Alternated deposition of bifunctional and tetra functional PEG molecules reacting through thiol/maleimide click chemistry is evaluated by quartz crystal microbalance. We first study parameters influencing the build-up process of such coatings and demonstrate the importance of (i) the nature of the first deposited layer, (ii) the PEG concentrations and (iii) the length of the PEG chains that appears to be the most significant parameter influencing film growth. The build-up process can be extended to a large variety of substrates like SiO2 or polymers by using an appropriate anchoring layer. Covalent functionalization of these nanogel films by proteins or enzymes is suited by modifying the biomolecules with thiol or maleimide groups and immobilizing them during the build-up process. Activity of the embedded enzymes can be maintained. Moreover ligands like biotin can be incorporated into the film and recognition by streptavidin can be modulated by playing with the number of PEG layers covering biotin. Compared to well-known PEG hydrogels, these new coatings are promising as they allow to (i) build thin nanometric coatings, (ii) finely control the amount of deposited PEG and (iii) organize the position of the embedded biomolecules inside the film layers.

Enhancement of gene delivery by an analogue of alpha-MSH in a receptor-independent fashion.

In order to transfect melanoma specifically by receptor-mediated endocytosis we prepared dioctadecyl aminoglycylspermine (lipospermine)--DNA complexes with [Nle(4),D-Phe(7)]-alpha-MSH(4--10), a pseudo-peptide analogue of alpha-melanocyte stimulating hormone (alpha-MSH) linked to a thiol-reactive phospholipid. With these complexes we obtained an up to 70-fold increase of transfection with B16-F1 melanoma cells. However when B16-G4F, an alpha-MSH receptor negative melanoma cell line was transfected, an up to 700-fold increased transfection efficiency was observed. The peptide hormone analogue was equally efficient when it was only mixed with lipospermine--DNA complexes without covalent coupling. In addition to melanoma cells we also obtained up to 30-fold increased transfection with BN cells (embryonic liver cells). Our data show that an alpha-MSH analogue increased transfection independently of the MSH receptor expression but reaches efficiencies approaching those obtained with peptides derived from viral fusion proteins. The absence of targeting of constructs containing [Nle(4),D-Phe(7)]-alpha-MSH(4-10) can probably be attributed due to the relatively modest number of MSH receptors at the surface of melanoma. We suggest, however, that the peptide hormone analogue used in this study has membrane-active properties and could be of interest as helper agent to enhance non-viral gene delivery presumably by endosomal-destabilizing properties.

Synthetic lipopeptides incorporated in liposomes: in vitro stimulation of the proliferation of murine splenocytes and in vivo induction of an immune response against a peptide antigen.

Amphiphilic lipopeptides, such as Pam3CysAlaGly and Pam3CysSerSer, were synthesized and incorporated into liposomes, and their ability to induce the proliferation of BALB/c mouse splenocyte was tested in vitro. When compared to monophosphoryl lipid A (MPL) the following potency order was found: liposomal lipopeptides > liposomal MPL > free (emulsified) lipopeptides. These results strongly depend on the size of the vesicles used: a mitogenic effect was observed only with lipopeptides incorporated within vesicles of diameter < or = 100 nm while lipopeptides in larger vesicles (diameter approximately 300 nm) gave no response. This may be related to the necessity for the liposome-associated lipopeptides to be endocytosed to reach putative intracellular targets. As immunoadjuvanticity seems to be linked to B-lymphocyte activation, the lipopeptides represent attractive alternatives to MPL for the realization of completely synthetic liposome-based peptide vaccine formulations. This was borne out by showing that Pam3CysAlaGly and Pam3CysSerSer, when incorporated in small unilamellar vesicles carrying a covalently conjugated synthetic peptide of sequence IRGERA, corresponding to an epitope of the C-terminal region of histone H3, were able to induce a potent and long-lasting immune response.

Osteoporosis as the sole presentation of bone marrow mastocytosis.

Three (young) adults with severe generalized osteopenia and vertebral compression fractures were studied. Extensive clinical and laboratory investigations were not contributory. Undecalcified bone biopsies demonstrated multiple mast cell granulomas in the marrow in two patients and numerous mast cells diffusely distributed throughout the bone marrow in the third patient. Mast cells may serve as a pathogenic agent in osteoporosis. Therefore, we conclude that isolated skeletal mastocytosis without clinical evidence of mast cell mediator release should be sought in the evaluation of a patient with unexplained severe bone loss.

Osteopenia, hematopoiesis, and bone remodelling in iliac crest and femoral biopsies: a prospective study of 102 cases of femoral neck fractures.

Cancellous microanatomy, bone remodelling, and hematopoietic tissue were studied in iliac crest and femoral neck biopsies taken intraoperatively in 102 consecutive patients with displaced intracapsular fractures. About half of the femoral biopsies had osteopenia, and most showed replacement of hematopoietic tissue by fat cells, with absence of osteoblasts and osteoclasts. In contrast, most of the iliac crest biopsies showed relatively less decrease in cancellous bone, while hematopoietic tissue and bone remodelling cells were present in approximately 75%. These results indicate that the iliac crest biopsy does not necessarily reflect cancellous microanatomy, hematopoietic tissue, and bone remodelling at other skeletal sites in any given patient.

Changes in trabecular bone, hematopoiesis and bone marrow vessels in aplastic anemia, primary osteoporosis, and old age: a comparative histomorphometric study.

Retrospective histologic analyses of bone biopsies and of post mortem samples from normal persons of different age groups, and of bone biopsies of age- and sex-matched groups of patients with primary osteoporosis and aplastic anemia show characteristic age dependent as well as pathologic changes including atrophy of osseous trabeculae and of hematopoiesis, and changes in the sinusoidal and arterial capillary compartments. These results indicate the possible role of a microvascular defect in the pathogenesis of osteoporosis and aplastic anemia.

Immunogenicity of new heterobifunctional cross-linking reagents used in the conjugation of synthetic peptides to liposomes.

We have investigated the immunogenicity of six thiol-reactive heterobifunctional cross-linking reagents that permit the conjugation of cysteine carrying peptides to the surface of liposome containing monophosphoryl lipid A. Such constructs elicit an immune response against short synthetic peptides and our aim was to find the least immunogenic linkers to limit potential carrier-induced epitopic suppression. For that purpose the properties of three new polyoxyethylene linkers of different lengths and thiol-reactive moieties (maleimide, bromoacetyl, dithiopyridine) were compared to known derivatives obtained by reacting the classical reagents SMPB and SPDP or N-succinimidyl bromoacetate with phosphatidylethanolamine. The least immunogenic linkers were the bromoacetate derivatives whereas those containing a maleimide group evoked a significant anti-linker immune response. In addition, using IRGERA as a model peptide, we found that all six liposomal constructs strongly elicited the production of anti-peptide IgG antibodies. This immune response was therefore independent of the length of the linkers (ranging between 0.3 and 1.6 nm) and of the nature of the linkage. between the peptide and the thiol-reactive moieties of the cross-linkers, i.e. stable thioether or bio-reducible disulfide bonds.

Morphologic classification of the myelodysplastic syndromes (MDS): combined utilization of bone marrow aspirates and trephine biopsies.

In a retrospective and prospective follow-up study from 1975 to 1991, bone marrow biopsies, aspirates and clinical features of 495 patients with MDS were investigated. Sections of undecalcified plastic embedded biopsies and smears of bone marrow aspirates were stained according to Giemsa. Bone marrows with MDS were characterized by three main categories of morphologic alterations: (1) cellular abnormalities, (2) architectural disorganization in the bone marrow and (3) stromal changes; the combined use of aspirates and trephine biopsies enabled a more reliable and accurate diagnosis of MDS than either one alone. The bone marrow findings fell into one of 7 subtypes, with the frequencies and median survivals in brackets: (1) MDS sideroblastic (19%, 62 months), (2) MDS megaloblastoid (13%, 56 months), (3) MDS proliferative (22%, 31 months), (4) MDS blastic (15%, 9 months), (5) MDS hypoplastic (15%, 26 months), (6) MDS fibrotic (6%, 29 months), and (7) MDS inflammatory (10%, 42 months). In follow-up studies patients with secondary MDS were excluded and the prognosis and subsequent evolution for each of the morphologic subtypes were evaluated. The conclusion is drawn that aspirates and trephine biopsies are complementary procedures and both are required for diagnosis, classification and decisions on current treatment modalities of patients with MDS.

Interferon-beta induced remission in a hairy cell leukemia patient resistant to interferon-alpha.

Two patients with advanced hairy cell leukemia and pancytopenia responded successfully to intramuscular injections of recombinant interferon-beta, with normalisation of blood counts. One of the patients had developed resistance to interferon-alpha. The in-vivo response was predicted by in-vitro studies showing a beneficial effect of IFN-beta on erythropoiesis in cell cultures derived from these patients.

Polyelectrolyte multilayer films with pegylated polypeptides as a new type of anti-microbial protection for biomaterials.

Adhesion of bacteria at the surface of implanted materials is the first step in microbial infection, leading to post-surgical complications. In order to reduce this adhesion, we show that poly(L-lysine)/poly(L-glutamic acid) (PLL/PGA) multilayers ending by several PLL/PGA-g-PEG bilayers can be used, PGA-g-PEG corresponding to PGA grafted by poly(ethylene glycol). Streaming potential and quartz crystal microbalance-dissipation measurements were used to characterize the buildup of these films. The multilayer films terminated by PGA and PGA-g-PEG were found to adsorb an extremely small amount of serum proteins as compared to a bare silica surface but the PGA ending films do not reduce bacterial adhesion. On the other hand, the adhesion of Escherichia coli bacteria is reduced by 72% on films ending by one (PLL/PGA-g-PEG) bilayer and by 92% for films ending by three (PLL/PGA-g-PEG) bilayers compared to bare substrate. Thus, our results show the ability of PGA-g-PEG to be inserted into multilayer films and to drastically reduce both protein adsorption and bacterial adhesion. This kind of anti-adhesive films represents a new and very simple method to coat any type of biomaterials for protection against bacterial adhesion and therefore limiting its pathological consequences.

Bone marrow histology in hairy cell leukemia. Identification of subtypes and their prognostic significance.

Bone marrow biopsy specimens taken on initial investigation of 134 patients with hairy cell leukemia (HCL) were processed into plastic, and sections cut at 3 microns were used for histologic and histomorphometric evaluation. Twenty-four clinical, 10 histologic, and four histomorphometric variables were tested for their prognostic significance. Bone marrow involvement was found in all cases, and on the basis of their morphology, the hairy cells were divided into three types: ovoid (47%), convoluted (37%), and indented (16%); this classification proved to be highly significant in the test statistics, with median survivals for the three groups of 56, 12, and 5 months, respectively. The characteristic nuclear features of the three types also were identified in smears of peripheral blood, in sections of spleen, and by electron microscopy. In addition, significant differences were found when the patients were grouped according to the tumor cell mass (volume %) in the biopsy sections, with median survivals of 55, 21, and 8 months noted for less than 20 vol%, 20-50 vol%, and greater than 50 vol%, respectively. Other factors with prognostic relevance were cytoplasmic inclusion bodies in the hairy cells (HC) and a number of clinical variables, including initial values of Hb and ESR, and platelets and monocytes in the peripheral blood. These results confirm that the bone marrow always is involved in HCL and its histology is diagnostic; that HCL may be classified according to the predominant neoplastic cell type; and that the patients may be staged on the basis of the tumor cell burden in the bone marrow biopsy.

Histologic classification and staging of multiple myeloma. A retrospective and prospective study of 674 cases.

Bone marrow biopsies of 674 patients with multiple myeloma (MM) were processed for diagnostic evaluation. Histologic variables were correlated with the clinical features to determine factors of value in predicting prognosis. Four of these were used to classify MM into six histologic types: Marschalko type; small cell type; cleaved type; polymorphous type; asynchronous type; and blastic type. These six types were subsequently combined into three prognostic grades: low, intermediate, and high, analogous to the malignant lymphomas. The quantity of plasma cell burden in the biopsy proved to be a useful criterion for histologic staging of MM, supplementing any clinical staging system in use. Both these parameters, grade and stage, provide information required for decisions on treatment modalities, while the effects of therapy can be monitored by sequential biopsies.

The bone marrow in aplastic anaemia: diagnostic and prognostic features.

Bone marrow preparations were examined from 80 patients with aplastic anaemia. The degree of cellularity varied greatly and in a third of the cases it was normal or even hypercellular at one site of aspiration. In the severely hypoplastic marrows lymphoid cells were predominant and this situation was associated with a worse prognosis. There was no correlation between marrow lymphoid cell content and blood lymphocyte count but there was an inverse relationship between blood lymphocyte count and marrow erythroblasts and a close direct relationship between the blood neutrophil count and marrow myeloid cell content. In all cases a proportion of the erythroblasts showed morphological abnormalities. These included especially megaloblastic changes and asynchrony of nuclear-cytoplasmic maturation. There were also binucleated cells, internuclear chromatin bridges, intercellular cytoplasmic connexions, nuclear degenerative changes, namely, blurred outlines, irregular shapes, budding and fragmentation, and atypical mitotic figures. These appearances illustrate the extent to which a qualitative defect of erythropoiesis occurs as part of the haematological pattern in aplastic anaemia, and in some cases dominates the bone marrow picture. Similar cytological features were found in all cases, including five patients with Fanconi's anaemia.

Bone biopsy in haematological disorders.

Bone marrow biopsies are now widely used in the investigation and follow-up of many diseases. Semi-thin sections of 8216 undecalcified biopsies of patients with haematological disorders were studied. Observations were made on the cytopenias and the myelodysplastic syndromes, the acute leukaemias the myeloproliferative disorders, Hodgkin's disease and the malignant lymphomas including multiple myeloma, hairy cell leukaemia and angioimmunoblastic lymphadenopathy. Bone marrow biopsies are essential for the differential diagnosis of most cytopenias and for the early recognition of fibrosis which most frequently occurred as a consequence of megakaryocytic proliferation in the myeloproliferative disorders. Different patterns of bone marrow involvement were found in the lymphoproliferative disorders and both their type and extent constituted factors of prognostic significance. A survey of the literature is given and the conclusion is drawn that bone marrow biopsies provide indispensible information for the diagnostic evaluation and the follow-up of patients with haematological disorders.

Plastic embedded core biopsy: a complementary approach to bone marrow aspiration for diagnosing acute myeloid leukaemia.

Bone marrow aspirates and biopsy specimens were taken at diagnosis from 51 patients with acute myeloid leukaemia (AML). The diagnosis was based on morphological and cytochemical analyses, and the leukaemias were classified by FAB criteria. A considerable difference was observed between the results of bone marrow aspirates and the findings of plastic-embedded bone marrow biopsy specimens, particularly in marrow cellularity, extent of blast cell infiltration, and cell type involved in the leukaemic process. The myelomonocytic cell type seemed to predominate in the sections. In four cases there was considerable marrow infiltration with maturing, but dysplastic, granulocytic cells in the sections, but not in the aspirate smears. Features of potential prognostic importance, such as bone marrow infiltration with inflammatory cells, were easily recognised and quantified in the sections. These results indicate that plastic embedded bone marrow biopsy sections complement the findings of bone marrow aspiration in the diagnosis of AML and may also provide information of independent prognostic importance that cannot be obtained by other means.

Lymphoproliferations in the bone marrow: identification and evolution, classification and staging.

Bone marrow biopsies from 3229 patients with lymphoproliferative disorders and 1156 patients with benign or reactive lymphoproliferations were investigated and criteria for distinguishing between them are given. Bone marrow involvement was found in 89% of multiple myeloma, 64% of non-Hodgkin's lymphomas and 8% of Hodgkin's disease. According to the predominant proliferative cell type there were five major entities in multiple myeloma and non-Hodgkin's lymphomas: (1) plasmacytic; (2) lymphocytic; (3) hairy cell; (4) immunocytic; (5) centrocytic. These were further classified into distinct subtypes each of which had independent prognostic significance. The mode of spread of the lymphoproliferative disorders in the bone marrow showed one of six architectural patterns, which together with the quantity of infiltration in the biopsy (reflecting the tumour cell burden) had significant predictive value. These results demonstrate the value of bone marrow biopsies in the identification, classification and staging of lymphoproliferative disorders, as well as in monitoring the course of disease and the response to therapy.

Working classification of chronic myeloproliferative disorders based on histological, haematological, and clinical findings.

Bone marrow biopsies of 850 patients with chronic myeloproliferative disorders were taken at initial diagnosis; and 169 sequential biopsies over periods of one to 188 months. Three micron sections of all biopsies were evaluated semiquantitatively with reference to the proliferating cell lines, anomalies of megakaryocytes, and fibrosis or osteosclerosis. Correlations between initial histological findings, clinical, haematological, and survival data were analysed statistically. The predominant cell lines distinguished the classical entities of polycythaemia vera, primary thrombocythaemia, and chronic myeloid leukaemia and correlated with their different prognoses, while megakaryocytes characterised subgroups that were prone to fibrotic or blastic transformation. Based on the initial histological, clinical, and haematological data analysed a working classification of chronic myeloproliferative disorders was proposed that permits recognition of both typical and atypical cases of chronic myeloproliferative disorders.

Bone scan and bone biopsy in the detection of skeletal metastases.

This study provides the first quantitative indication of the limits of sensitivity of a bone scan with technetium (99Tc-MDP) in detecting skeletal metastases and thereby also helps to explain the fact that bone scans may be negative when metastases are present in the bone marrow. Since 99Tc-MDP remains the least noxious and most widely used isotope for bone scanning, these results have direct clinical relevance in the evaluation of patients with solid tumors and possible metastatic spread.