Lactococcus strains treated with heat and hen-egg-white lysozyme induce abundant interleukin-12 production by J774.1 macrophages and murine spleen cells.

The IL-12-inducing ability of lactic acid bacteria could be a critical index of immunomodulatory activity, especially in promoting T-helper-1 responses and in suppressing T-helper-2-mediated allergic responses. We aimed to develop a simple method for enhancing the IL-12-inducing ability of bacteria. We examined the in vitro effects of strains of lysozyme-modified Lactococcus (ML-LYS), prepared by heat treatment of the Lactococcus strain in the presence of lysozyme, on the ability of mouse macrophage-like J774.1 cells and spleen cells to produce IL-12. An IL-12-inducing ability greater than that of heat-killed bacteria was shown by 41 of 46 ML-LYS strains in J774.1 cells and by all 46 ML-LYS strains in mouse spleen cells. In contrast, bacteria modified by α-lactalbumin, ß-lactoglobulin, or ovalbumin did not enhance IL-12 production in J774.1 cells. Microscopically, ML-LYS showed stronger resistance to lysozyme and macrophage digestion than did heat-killed bacteria or the other modified bacteria. Addition of chitotriose, a lysozyme inhibitor, enhanced IL-12 production by J774.1 cells stimulated with heat-killed bacteria. Therefore, enhancement of resistance to lysozyme may be a key factor in the strong IL-12-inducing ability of ML-LYS. These findings have important implications for the design of dairy products that have an immunomodulatory effect using the modified bacteria.

Spontaneous uterine leiomyosarcoma in a golden hamster (Mesocrietus auratus).

Uterine leiomyosarcoma occurring spontaneously in a domestic golden hamster was examined histologically and immunohistochemically. The histological findings for this tumour were similar to those for leiomyosarcomas described in other species. Immunohistochemical examination demonstrated the positivity of neoplastic cells with alpha-smooth muscle actin and desmin. From the results mentioned above, the tumour of this case was revealed to be of smooth muscle origin. To our knowledge, this is the first report of a uterine leiomyosarcoma in domestic golden hamsters.

Successful transcutaneous arterial embolization of a giant hemangioma associated with high-output cardiac failure and Kasabach-Merritt syndrome in a neonate: a case report.

We describe the case of a patient with a neonatal giant cutaneous hemangioma with high-output cardiac failure and Kasabach-Merritt syndrome and successfully treated with transcutaneous arterial embolization aimed at controlling severe congestive heart failure and consumption coagulopathy. A patient was admitted to the neonatal care unit on the first day of age because of a large hemangioma on his right lateral chest wall and respiratory distress, associated with cardiac failure resulting from arteriovenous shunting. On the second day of age the platelet count decreased to 5.7 x 10(4)/microliter and fibrinogen level was 85 mg/dl. The values of prothrombin time and activated partial thromboplastin time were prolonged. Intravenous predonisone therapy was started immediately, but bleeding tendency was getting worse and the evidence of congestive heart failure persisted. On the third day the patient then underwent embolization of feeding arteries with microcoils. The cardiac failure and thrombocytopenic coagulopathy had improved significantly without complications. We conclude that transcutaneous arterial embolization is an effective and safe treatment in this neonate and should be considered for the treatment of control high-output cardiac failure and coagulopathy in infants with hemangioma and Kasabach-Merritt syndrome.

Effects of cis-diamminedichloroplatinum(II) on Escherichia coli and bacteriophage systems.

The effects of cis-diamminedichloroplatinum(II) (cisplatin) on Escherichia coli cells and bacteriophages were investigated. The bacteriocidal effect of cisplatin was stronger on uvrA or recA mutants than on wild type cells. The drug, like UV, induced prophage development in lysogenic bacteria. Host cell reactivation of alpha3 replicative form (RF) I DNA treated with cisplatin in vitro was more efficient in wild type or recA cells than in uvrA host. When wild type cells were exposed to cisplatin, decay of the host's capacity to sustain the viral multiplication proceeded nearly in parallel with the loss of colony-forming ability, whereas the capacity of uvrA mutant was much more resistant to the drug, as compared with the viability. In the DNA preparation from cisplatin-treated alpha3-infected wild type cells, RF II was deficient, but the RF I molecules extracted from the cells were moderately infective. The microvirid gene A protein, required for RF I-->RF II conversion, was hardly detectable in wild type cells exposed to cisplatin. The possible relationship between uvr+-dependent repair and synthesis of the viral protein is discussed.

Effects of synthetic hydroxy isothiocyanates on microbial systems.

Hydroxy isothiocyanates (ITCs), including some new derivatives of naturally occurring compounds, were synthesized and their minimum inhibitory, minimum fungicidal, and minimum bactericidal concentrations for Aspergillus niger, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli were estimated. These compounds were strongly antimicrobial; for example, 2-(4-hydroxyphenyl) ethyl ITC inhibited growth of all strains examined at concentrations of 7.8 to 15.6 micrograms/ml. The ATP concentration in E. coli was markedly reduced when cells were treated with 2-(4-hydroxyphenyl)ethyl ITC. Inhibition of the growth of E. coli by 2-(4-hydroxyphenyl)ethyl ITC was decreased in the presence of cysteine. Streptolysin S production in washed cells of Streptococcus equisimilis was extremely sensitive to this ITC derivative and this inhibition also was counteracted by cysteine. The results showed that the ITC compounds had antimicrobial effects by blocking sulfhydryl groups.

Mouse germinal center B cells with the xid mutation retain responsiveness to antimouse CD40 antibodies but diminish IL-5 responsiveness.

The germinal center (GC) develops in secondary lymphoid tissues in response to thymus-dependent (TD) antigens. To investigate the molecular mechanism of B cell differentiation in GC, we enriched GC B cells from spleen of TD antigen-immunized wild-type and X-linked immunodeficient (XID) mice, and examined the differentiation of GC B cells into antigen-specific IgG1 antibody-forming cells (AFC) in response to anti-CD40 mAb and cytokines. A significant proportion of freshly purified GC B cells expressed receptors for IL-4 and IL-5. Anti-CD40 mAb sustained the viability of GC B cells and IL-4 co-operated with anti-CD40 mAb for further enhancement of the cell viability. Anti-CD40 mAb and IL-4 were essential for inducing differentiation of GC B cells into antigen-specific IgG1-AFC and IL-5 efficiently enhanced their differentiation. GC B cells with the xid mutation responded for proliferation to CD40 ligation to a lesser extent and for the IgG1-AFC response to anti-CD40 mAb together with IL-4, but they showed impaired responsiveness to IL-5, regardless of enhanced expression of IL-5R in response to anti-CD40 mAb and IL-4. These results suggest that anti-CD40 mAb, IL-4 and IL-5 play a critical role in the differentiation of mouse GC B cells. The GC B cells from XID mice show a functional defect with respect to IL-5-mediated differentiation.

The N-terminal sequence of Lactococcus lactis phosphoglucose isomerase purified by affinity chromatography differs from the other species.

A specific monoclonal antibody, M3A, was produced to rapidly purify Lactococcus lactis phosphoglucose isomerase (PGI) for amino acid sequence analysis. M3A recognized the Lac. lactis PGI specifically and sensitively with both enzyme-linked immunosorbent assay and Western blot analysis. The enzyme was rapidly purified to a specific activity of 21.8 U/mg with a yield of 20% by a three-step procedure, including M3A-bound Sepharose chromatography. The specific activity of PGI was increased about 64.1-fold from the cell lysate. The molecular mass of Lac. lactis PGI was estimated to be about 50 kDa by SDS-PAGE. The N-terminal amino acid sequence of Lac. lactis PGI exhibited no significant similarity to other PGIs, except for a 52.6% identity to Bacillus stearothermophilus PGI A and PGI B. These results suggest that there might be some molecular types of PGI.

CD43 expression in a B cell lymphoma, WEHI 231, reduces susceptibility to G1 arrest and extends survival in culture upon serum depletion.

CD43 is a major surface sialoprotein on hemopoietic cells, whose extracellular domain is heavily O-glycosylated. The functional role of CD43 in the hemopoietic system is not fully understood; however, it has been suggested that CD43 may have a role in cell-cell repulsion and in modifying T cell proliferation and activation. CD43 is expressed in immature B cells in the bone marrow, but not by peripheral B cells, except for B-1 B cells and plasma cells. To analyze the biological effect of CD43 in B-lineage cells, we transfected mouse CD43 cDNA into a CD43- B cell lymphoma, WEHI 231, and the growth and survival in culture were compared to those of a parental cell line, human CD8 transfectants, and CD43- revertants established from CD43+ clones. We observed that CD43 expression supported cell growth in culture upon serum reduction, whereas growth of CD43- cell lines was barely detected under this condition. CD43- cell lines accumulated in G1 phase of the cell cycle, and the numbers of viable cells were greatly reduced during culture upon serum depletion, whereas expression of CD43 reduced the susceptibility to G1 arrest and temporarily retarded the apoptotic process, which, in turn, resulted in an increase and maintenance of the number of viable cells in culture. The results suggest that CD43 may have some role in the survival and expansion of B-lineage cells. The biological effect of CD43 was initiated without stimulation by cross-linking and was significantly impaired by replacement of the extracellular domain by the human CD8 extracellular domain. The basis of these regulatory processes is discussed.

Expression of Vpre-B3 (8HS-20) molecules by alternative RNA processing.

In pre-B cells, mu chains are expressed in association with "surrogate' L chains encoded by the lambda 5 and Vpre-B1 genes. In addition to their association with lambda 5 and Vpre-B1, mu chains in pre-B cells are associated with the products of the Vpre-B3 gene (formerly designated 8HS-20), which display a distinct association with mu chains and biochemical properties in terms of mol. wt, pI value and glycosylation. However, the mechanism of the generation of Vpre-B3 isoforms has been unknown. The present study indicates that the Vpre-B3 gene transcript underwent alternative RNA processing in normal B cells, in a pre-B cell lymphoma and in a mature B cell lymphoma, WEHI 231, that was transfected with the Vpre-B3 genomic clone. Vpre-B3 isoforms were expressed in a WEHI 231 cell line transfected with the Vpre-B3 genomic clone, comparable in biochemical nature to those expressed in a pre-B cell lymphoma. In contrast, expression of one of the isoforms was missing in a cell line transfected with the Vpre-B3 cDNA clone. These results suggest that Vpre-B3 isoforms with distinct biochemical characteristics are derived from alternatively processed Vpre-B3 mRNA.

The virion proteins encoded by bacteriophage phi K and its host-range mutant phi KhT: host-range determination and DNA binding properties.

The microvirid phage phi K, specific for Escherichia coli K12, contains a circular single-stranded (SS) DNA in the icosahedral virion, which comprises four phage gene products, F (capsid), G (major spike), H (minor spike), and J (core). phi KhT, a host-range mutant of phi K, can grow on E. coli C and B, besides K12, and is more thermosensitive than the parental phage phi K. Sequencing analysis revealed that the genome of phi K and phi KhT consists of 6,089 nucleotides (nt), and codes for eleven genes, whose sequences are similar to those of alpha 3, phi X174, and G4 infective to strain C. In phi KhT, two nt had changed: one is in the gene G, resulting in replacement of the 75th codon Ala with Ser, and the other is at 67th codon of the gene H: Val to Ala. Chemically synthesized gene J protein composed of 23 amino acids (aa) binds to phi K SS DNA more tightly than and preferentially over the host E. coli SS-DNA-binding protein (SSB). These results indicate that the two spike proteins G and H are involved in the determination of phi K host-range, and support a model in which the gene J protein functions in packaging the viral SS DNA into the virion vesicle.

Cerebral hemodynamics on near-infrared spectroscopy in hypoxia and ischemia in young animal studies.

Using near-infrared spectroscopy the changes of intracranial oxyhemoglobin, deoxyhemoglobin, total hemoglobin and cytochrome aa3, which show the progression of intracranial oxygenation, hemodynamics and cell metabolism, were recorded during prolonged partial hypoxia induced by carbon dioxide (CO2) and nitrogen (N2), ischemia induced by hyperventilation, and hypoxia during hypoglycemia in neonatal and young rabbits. The reduction of cytochrome aa3 during the terminal stage of CO2-induced prolonged hypoxia, hyperventilation and hypoxia in hypoglycemia suggests that the redox state of cytochrome aa3 will be changed by several combined factors such as oxygen delivery, ATP demand and substrate (glucose) delivery.

The fetal thymus stores immature hemopoietic cells capable of differentiating into non-T lineage cells constituting the thymus stromal element.

Immature hemopoietic cell lines were established by transforming fetal thymocytes in vitro with a ts mutant of Abelson murine leukemia virus. They are positive for c-kit and IL-2R alpha but negative for lineage specific markers. Their TCR and Ig heavy chain genes are in germline configuration, and are expressed as germline gene transcripts. When these cell lines were stimulated in vitro with IL-1 their morphology changed into that of typical macrophages (M phi). Subsequent analysis of a particular clone, which displayed the morphological change at the highest efficiency among established cell lines, indicated that the clone possesses the capacity to differentiate into I-A-M phi capable of secreting several cytokines, and supporting the proliferation of fetal and adult thymocytes in vitro. If their surface markers are considered, their normal counterparts would be present in a minor subset of CD4-CD8- double-negative cells in the thymus in early development. The results raise the possibility that the thymic organ at an early stage of development stores immature hemopoietic cells capable of differentiating into a non-T lineage constituting the thymic stromal elements.

Heavy chain variable (VH) region diversity generated by VH gene replacement in the progeny of a single precursor cell transformed with a temperature-sensitive mutant of Abelson murine leukemia virus.

Sequence analysis of a large number of DNA clones containing a functional heavy chain variable, diversity, and joining (VHDJH) complex generated by VH to VHDJH joining (VH gene replacement) in the progeny derived from a common precursor cell transformed with a temperature-sensitive (ts) Abelson murine leukemia virus (A-MuLV) indicates that endogenous VH gene replacement in vitro generates immunoglobulin gene joints distinct from those generated by the usual VH to DJH joining. Such joints keep the pentamer CAAGA at the 3' end of the donor VH segment and lack a recognizable D segment, as can be seen also in vivo. The results suggest that VH gene replacement participates in generating VH region diversity in vivo, as previously postulated. During the joining process, a unique VH gene was selected in all progeny cells, together with a single A nucleotide dominantly added to the junctional boundaries. The basis of these regulatory processes is discussed.

Two types of mu chain complexes are expressed during differentiation from pre-B to mature B cells.

Immunoglobulin mu chains synthesized in murine pre-B cells are known to be associated with surrogate light chains designated as omega (omega), iota (iota) and B34. In addition to these molecules, we identified the complexes of polypeptides (50, 40, 27 and 15.5 kd) associated with surface or intracellular mu chains of pre-B cell lines. Most of these polypeptides were continuously synthesized and associated with mu chains in virgin B cells lines, although some of them scarcely bound to the mu kappa dimer or mu 2 kappa 2 tetramer concomitantly present in the same clone or population. However, in mature B cells they were no longer detectable except B34. Cross-linking of micron chains on the surface of pre-B cells resulted in an increase in intracellular free Ca2+, indicating that the micron chain complex on the surface of pre-B cell lines acted as a signal transduction molecule. However, the receptor cross-linkage of pre-B cell lines did not induce the increased inositol phospholipid metabolism usually observed in virgin and mature B cell lines. These results suggest that, during the differentiation from pre-B to mature B cells, the cells express two types of mu chain complexes which exhibit different structures as a whole and possess different signal transducing capacities.

A case of leiomyosarcoma associated with humoral hypercalcemia of malignancy: demonstration of biological and immunological activities of parathyroid hormone-related protein in the tumor extract.

Hypercalcemia occurred in a patient with leiomyosarcoma when multiple lung metastases developed. Despite normal plasma parathyroid hormone (PTH) levels and low 1,25-dihydroxyvitamin D, this hypercalcemic patient had a marked hypercalciuria and phosphaturia associated with an increased excretion of nephrogenous cyclic AMP (NcAMP). Administration of cisplatin ameliorated both the hypercalcemia and hypercalciuria without any reduction in tumor size of NcAMP excretion. Terminally, acute pancreatitis occurred producing a profound hypocalcemia. In the extract of tumor tissue obtained post mortem, bioactivity stimulating the generation of cyclic AMP in osteogenic cells was demonstrated along with the immunoreactive PTH-related protein (PTH-rP). the first report of a solid non-epithelial malignancy producing PTH-rP and associated with humoral hypercalcemia of malignancy. The hypercalcemia in this case caused acute pancreatitis, which led to a profound hypocalcemia.

B cell precursors are present in the thymus during early development.

An in vitro system for transforming immature lymphoid cells present in the thymus at early development has been established. By phenotype analysis of the transformants obtained, we observed that B cell precursors, susceptible to Abelson murine leukemia virus (A-MuLV)- or Harvey murine sarcoma virus (H-MuSV)-induced lymphogenesis, were present at high frequency in the fetal thymus of BALB/c mice. These precursors recolonized alymphoid thymus lobes in vitro, as do T cell precursors. It was further observed that B precursors in the fetal liver were also capable of recolonizing alymphoid thymus lobes and were stored in a thymic environment. These results suggest that stroma cells of the fetal thymus may possess the capacity to support the growth of B precursors. On the other hand, B cell precursors sensitive to the viral transformation were undetectable in the fetal thymus of C57BL/6, although immunohistochemical analysis suggested their presence. However, in the fetal liver of the same strain, B precursors recolonizing alymphoid thymus in vitro were sensitive to the viral transformation. Based on these results, we will discuss both the role and fate of thymic B precursors. In addition, we also obtained T cell lymphomas at different stages of differentiation from the fetal thymus of C57BL/6 infected with A-MuLV or H-MuSV. These data indicate the usefulness of our system in establishing cell lines derived from intrathymic lymphogenesis at early development.

Antitumor activity and neutrophil-selective hematopoietic toxicity of busulfan analogs in mice.

The antitumor activity and hematopoietic toxicity of two busulfan analogs were evaluated in comparison with those of busulfan. Although a program of five daily ip treatments with busulfan was not effective in treating sarcoma 180-bearing mice, a fluorine-containing busulfan analog, 1,4-butanediol di-2,2,2-trifluoroethanesulfonate (BFS), and a water-soluble analog, 1,4-butanediol diisethionate (BIT), were significantly effective when given on the same schedule. Busulfan did not appreciably prolong the life span of either P388- or Meth A-bearing mice, whereas BFS and BIT produced significant increases in the life span. It is worth noting that both the analogs were definitely less toxic to the host mice than busulfan. All the drugs examined exhibited suppressive effects on the counts of total WBCs, neutrophils, and lymphocytes. Relative toxicity toward neutrophils versus lymphocytes was increased significantly in the BFS and BIT treatments compared with busulfan treatment. It seems that the toxicity of busulfan in host mice might be due to unidentified side effects other than bone marrow suppression. These results suggest that BFS and BIT could be improved substitutes for busulfan.