Endogenous tissue engineering: PTH therapy for skeletal repair

Based on its proven anabolic effects on bone in osteoporosis patients, recombinant parathyroid hormone (PTH_1-34) has been evaluated as a potential therapy for skeletal repair. In animals, the effect of PTH_1-34 has been investigated in various skeletal repair models such as fractures, allografting, spinal arthrodesis and distraction osteogenesis. These studies have demonstrated that intermittent PTH_1-34 treatment enhances and accelerates the skeletal repair process via a number of mechanisms, which include effects on mesenchymal stem cells, angiogenesis, chondrogenesis, bone formation and resorption. Furthermore, PTH_1-34 has been shown to enhance bone repair in challenged animal models of aging, inflammatory arthritis and glucocorticoid-induced bone loss. This pre-clinical success has led to off-label clinical use and a number of case reports documenting PTH_1-34 treatment of delayed-unions and non-unions have been published. Although a recently completed phase 2 clinical trial of PTH_1-34 treatment of patients with radius fracture has failed to achieve its primary outcome, largely because of effective healing in the placebo group, several secondary outcomes are statistically significant, highlighting important issues concerning the appropriate patient population for PTH_1-34 therapy in skeletal repair. Here, we review our current knowledge of the effects of PTH_1-34 therapy for bone healing, enumerate several critical unresolved issues (e.g., appropriate dosing regimen and indications) and discuss the long-term potential of this drug as an adjuvant for endogenous tissue engineering.     

Antibody-induced association between discrete regions of HLA class I and II antigens

The anti-HLA-DR + DP monoclonal antibody (MoAb) CR11-462 was unexpectedly found to cross-inhibit the binding to B lymphoid cells of the anti-HLA Class I MoAb CR10-215 and CR11-115. The latter two antibodies recognized the same or spatially close antigenic determinant. The cross-blocking of anti-HLA Class I MoAb CR10-215 and CR11-115 by MoAb CR11-462 reflects neither its contamination by anti-HLA Class I antibodies nor its cross-reactivity with HLA Class I antigens. On the other hand, the cross-blocking appears to reflect redistribution of HLA Class II antigens by the MoAb CR11-462, since the MoAb CR10-215 and CR11-115 are not susceptible to blocking when lymphoid cells are treated with 0.025% glutaraldehyde or are coated with Fab' fragments of the MoAb CR11-462. Furthermore, immunoprecipitates from B lymphoid cells preincubated with the MoAb CR11-462 before solubilization contain HLA Class I antigens. Therefore, these results have shown for the first time an antibody-induced association between discrete regions of HLA Class I and Class II antigens on the membrane of B lymphoid cells.     

The establishment of IL-2 producing cells by genetic engineering

Expression plasmids containing human interleukin-2(IL-2) cDNA under the control of viral promoters (SV40 early region, MuLV LTR, HTLV-I LTR, and ASV (Y73) LTR) were introduced into TK- mouse L cells and human FL cells to establish IL-2 producing cells. The highest levels of IL-2 producing clones were obtained in TK+ mouse L cells transformed with a recombinant plasmid having MuLV LTR as a promoter, whereas transformed cells of human FL cells (G418r) were revealed to produce IL-2 at the highest level when the cells were transfected with a plasmid containing HTLV LTR as a promoter. These results suggest that these promoter/enhancer regions possess different cell specificities in gene expression. To obtain higher levels of IL-2 production using gene amplification, the hybrid plasmids containing the hamster DHFR and human IL-2 genes were constructed and transfected into DHFR- CHO cells. DHFR+ colonies produced IL-2 at about the same level as that produced by TK+ L cells transformed with the recombinants containing MuLV LTR. Selection of methotrexate-resistant cells resulted in a 5- to 30-fold increase of IL-2 production. These cells produced IL-2 stably for at least 3 months, even in the absence of methotrexate.     

Decrease of palmitoyl-CoA elongation in platelets and leukocytes in the patient of hereditary methemoglobinemia associated with mental retardation

Effect of the deficiency of NADH-cytochrome b5 reductase on fatty acid elongation was studied in the platelets and leukocytes taken from a patient of hereditary methemoglobinemia associated with mental retardation. The activity of fatty acid elongation was determined by measuring the incorporation of [2-14C]malonyl-CoA into palmitoyl-CoA. The de novo biosynthesis of fatty acids was blocked by the addition of phosphotransacetylase, and the elongation system could be assayed in the homogenates separated from de novo biosynthesis. As compared to normal subjects approximately 40% decrease of fatty acid elongation was observed both in the platelets and leukocytes from the patient.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Rapid formation of myometrial gap junctions during parturition in the unilaterally implanted rat uterus

Summary In uterine smooth muscles, gap junction plaques rapidly form during the final stages of gestation. To investigate the related mechanisms, regional differences in myometrial gap junction development in rat uterus were examined quantitatively during delivery, using thin-section and freeze-fracture techniques in combination with light- and electron microscopy.Examination of implanted and nonimplanted horns in the unilaterally ligated rat bicornuate uteri, revealed no differences in the occurrence of gap junction plaques, but after 2 to 4 pups had been delivered, the contracted segments contained more gap junction plaques than did noncontracted segments examined immediately before delivery. In all segments, gap junctions were found more frequently in the circular muscle layers than in the longitudinal muscle layers. Gap junctions ranged in size from 0.002 m² to 0.52 m², but two-thirds were less than 0.1 m². The frequency of small gap junction plaques (less than 0.1 m²) was higher in the noncontracted segment.These results suggest that gap junctions are dynamic structures, and that their formation is controlled not only by general hormonal factors, possibly involved in gap junction increases in the myometrium before delivery, but also by local factors, possibly related to the contraction, that may accelerate an increase in gap junction formation during delivery.     

Splenic outer periarterial lymphoid sheath (PALS): An immunoproliferative microenvironment constituted by antigen-laden marginal metallophils and ED2-positive macrophages in the rat

Summary In an attempt to reveal the role of antigen-laden marginal metallophil (MM) and other macrophages in the intrasplenic immune response of a specific B-cell lineage to a thymus-independent type-2 antigen (Ficoll conjugated with fluorescein isothiocyanate), simultaneous immuno-histological observations of the involved cells were performed in the rat. By newly established methods of double or triple immunostainings, time-kinetics of the following parameters were studied and compared: (1) the antigen, (2) the specific antibody-forming cells (AFC) directed to the fluorescein-isothiocyanate determinant, (3) proliferating cells labeled with 5-bromo-2-deoxyuridine (BrdU), and (4) macrophage subpopulations recognized by monoclonal antibodies (ED2 and ED3). The antigen localized stably not only in the marginal-zone macrophages but also in the MM except around the follicular area. The increase of BrdU-positive cells was observed from day 2 up to day 4 after antigen injection mostly in the periphery of the periarterial lymphoid sheath (outer PALS), which indicated antigen-induced proliferation. As a novel finding, the majority of AFC, both BrdU-positive and -negative, were either closely associated with the antigen-laden MM, or forming cell clusters with ED2-positive macrophages in the outer PALS. In contrast, there were very few AFC in juxtaposition to antigen-free MM in the follicular area or the antigen-laden marginal zone macrophages. The results led to the proposal of a hypothesis that the antigen-laden MM together with ED2-positive macrophages constitute an immunoproliferative microenvironment for the plasmacellular reaction by accumulating the antigen-specific B-cell lineage and promoting these cells to differentiate into the AFC and to proliferate in the outer PALS.Abbreviations AFC specific antibody-forming cells - BrdU 5-bromo-2-deoxyuridine - Fic-F FITC-conjugated Ficoll - FITC fluorescein isothiocyanate - HRP horseradish peroxidase - MM marginal metallophils - MZ marginal zone - PALS periarterial lymphoid sheath - PBS phosphate-buffered saline - TI2 thymus-independent type-2     

Identifying the fluorescent body (F-body) with quinacrine mustard staining of canine

The fluorescent body (F-body) was identified with quinacrine mustard (Q-M) staining in spermatozoon and lymphocyte of canine. Well washed sperm suspension was treated with protease (125 mg/ml) or dispase (2000p. u./ml) and staining with Q-M (final dilution 50 micrograms/ml) for 15 min to 24 hr at 37 degrees C. The lymphocyte cultures from whole blood were prepared as routine human investigation. The chromosomal preparation made by air dry method was stained with Q-M (final dilution 0.5 to 50 micrograms/ml) after pretreatment of enzyme digestion. The examination using a reflected fluorescent microscope revealed that the same F-body in human was present in both spermatozoon (20.1-39.7%) and interphase of lymphocyte (0.37.2%) of male origin.