Showing papers on "In vitro published in 1988"

Dextran sulfate suppression of viruses in the HIV family: inhibition of virion binding to CD4+ cells

Abstract: The first step in the infection of human T lymphocytes by human immunodeficiency virus type 1 (HIV-1) is attachment to the target cell receptor, the CD4 antigen. This step may be vulnerable to attack by antibodies, chemicals, or small peptides. Dextran sulfate (molecular weight approximately 8000), which has been given to patients as an anticoagulant or antilipemic agent for more than two decades, was found to block the binding of virions to various target T lymphocytes, inhibit syncytia formation, and exert a potent inhibitory effect against HIV-1 in vitro at concentrations that may be clinically attainable in human beings. This drug also suppressed the replication of HIV-2 in vitro. These observations could have theoretical and clinical implications in the strategy to develop drugs against HIV types 1 and 2.

IFN-gamma stimulates IgG2a secretion by murine B cells stimulated with bacterial lipopolysaccharide.

Abstract: rIFN-gamma strikingly enhances the secretion of IgG2a by murine splenic B cells stimulated with bacterial LPS in vitro and concomitantly suppresses the production of IgG3, IgG1, IgG2b, and IgE while sparing IgM secretion. IFN-gamma stimulates highly purified B cell populations to secrete IgG2a, strongly suggesting that it acts directly on B cells. It increases the frequency of precursors of IgG2a-expressing soft agar colonies and enhances the number of IgG2a+ cells in colonies indicating that it both increases the frequency of precursors of IgG2a+ cells and enhances the number of IgG2a+ daughter cells emerging from each precursor. IFN-gamma completes its action within the first 24 to 48 h of a 6-day culture with LPS and its addition cannot be delayed beyond the first 48 h. Preincubation of resting B cells in the presence of IFN-gamma leads to a time dependent increase, up to 42 h, in IgG2a secretion upon subsequent addition of LPS. IFN-gamma can exert this action on resting B cells that have been selected for absence of membrane IgG expression by cell sorting. The promotion of IgG2a secretion appears to be a specific property of IFN-gamma in that IFN-alpha, IFN-beta, IL-1, IL-2, IL-3, IL-4, IL-5, granulocyte-macrophage-CSF, granulocyte-CSF, and CSF-1 fail to enhance IgG2a secretion by LPS-stimulated B cells.

A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures

Abstract: A literature review was conducted using original papers published during 1964-1985 on the in vitro clastogenicity of chemical substances. Results of tests on 951 chemical substances were abstracted from over 240 reports to form the database. The evaluation of these data relied on each author's original conclusion on a positive or negative outcome. Of these 951 substances, 447 (47%) were consistently positive either with or without activation; 417 (44%) were negative in the direct test but not tested with metabolic activation systems; 4 were negative but tested only with activation; and 30 (3%) were clearly negative both with and without activation. The remaining 53 substances gave variable results when tested under different experimental protocols or in different cell types, but were positive in at least one test. Although discrepant results were found associated with some cell types, the addition of metabolic activation systems tended to eliminate such variability. No one cell appeared to be superior in response to all clastogens. For screening purposes, the choice of cell may thus depend more on the general usefulness and reliability of a cell type than on a strong response to a particular chemical. However, the use of a suitable metabolic activation system does appear to be of critical importance. The concentration at which clastogenic effects were detected varied extensively for different test substances, ranging from a minimum of 4.3 X 10(-8) to 6.9 X 10(2) mM. Possible mechanisms of action for substances active at only high levels are discussed, but no satisfactory explanation is available at this time. The relevance of tests conducted at concentrations high enough to alter significantly the osmolarity and other culture conditions is considered, and caution urged in the interpretation of test results obtained under physiologically stressful conditions. The clastogenic potential was compared quantitatively using an index of effective concentration (D20) and one which estimates the number of cells with exchange aberrations expected per mg/ml (TR) for data obtained by using a uniform protocol and cultures of Chinese hamster lung (CHL) cells. Both values were distributed over a wide range, demonstrating the variety of genotoxic potential in chemicals. In general, a substance which was active at only high concentrations produced fewer exchange-type aberrations. In vivo activity, as measured by tumourigenic effect and formation of micronuclei in bone marrow, tended to be greater for substances with a D20 below 10(-2) mg/ml and a TR value over 10(3).(ABSTRACT TRUNCATED AT 400 WORDS)

Therapeutic attack of hypoxic cells of solid tumors: presidential address

Abstract: Hypoxic cells of solid tumors are relatively resistant to therapeutic assault. Studies have demonstrated that oxygen-deficient tumor cells exist in an environment conducive to reductive reactions making hypoxic cells particularly sensitive to bioreductive alkylating agents. Mitomycin C, the prototype bioreductive alkylating agent available for clinical use, is capable of preferentially killing oxygen-deficient cells both in vitro and in vivo. This phenomenon is at least in part the result of differences in the uptake and metabolism of mitomycin C by hypoxic and oxygenated tumor cells, with the ultimate critical lesion being the cross-linking of DNA by the mitomycin antibiotic. The combination of mitomycin C with X-irradiation, to attack hypoxic and oxygenated tumor cell populations, respectively, has led to enhanced antitumor effects in mice bearing solid tumor implants and in patients with cancer of the head and neck. More efficacious kill of hypoxic tumor cells may be possible by the use of dicoumarol in combination with mitomycin or by the use of the related antibiotic porfiromycin. The findings support the use of an agent with specificity for hypoxic tumor cells in potentially curative regimens for solid tumors.

Normal mouse peritoneum contains a large population of Ly-1+ (CD5) B cells that recognize phosphatidyl choline. Relationship to cells that secrete hemolytic antibody specific for autologous erythrocytes.

Abstract: We have found that, in the peritoneums of normal adult mice, 5-15% of lymphocytes bind a fluorescent liposome probe. In ontogeny, cells with this specificity were shown to appear by 8 d after birth, and increase to the adult frequency by 2-3 wk. Some older mice contain an expanded population of these cells. We have shown that liposome binding occurs by cell surface IgM recognizing the common membrane phospholipid, phosphatidyl choline (PtC). Virtually all of these PtC-specific cells bear the cell surface marker Ly-1. Our results indicate that roughly 1 in 10 peritoneal Ly-1+ B cells has this single specificity. We have found that the precursors to all the cells that form plaques on protease-treated autologous erythrocytes (BrMRBC) are included in the PtC-specific population and can be isolated by FACS. We believe this is the first report of sorting large numbers of B cells with a single antigen specificity from normal, unimmunized animals. This method will allow for in vitro and in vivo studies of differentiative and proliferative properties of Ly-1+ B cells, which may help define their role in development and disease.

Multiple Human Progesterone Receptor Messenger Ribonucleic Acids and Their Autoregulation by Progestin Agonists and Antagonists in Breast Cancer Cells

Abstract: We have used AB-52, a monoclonal antibody which recognizes both the A (94,000 daltons) and B (120,000 daltons) proteins of human progesterone receptors (hPR), and hPR-50, a PR complementary DNA probe isolated from a T47D-pcD library, to study the structure and hormonal regulation of the hPR mRNAs and proteins in human breast cancer cells. RNA blot hybridization analysis of poly(A+) RNA shows that T47DCO, an estrogen resistant human breast tumor cell line in which PR are constitutively expressed, contain at least six PR mRNAs ranging in size from 2.5 to 11.4 kilobases. All six are mature cytoplasmic messages that are also present in normal human endometrium and in PR-positive MCF-7 breast cancer cells, but not in PR-negative cells. Using hPR-50 RNA synthesized in vitro as a 1.3 kilobase standard, we calculate that MCF-7 cells contain approximately 16 message molecules per cell which are increased to approximately 45 by estradiol treatment; T47DCO cells contain approximately 90 message molecules per cell co...

Investigation of the biological effects of anti-cell adhesive synthetic peptides that inhibit experimental metastasis of B16-F10 murine melanoma cells.

Abstract: The experimental metastasis of B16-F10 murine melanoma cells is blocked by the anti-cell adhesive pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) derived from the central cell-binding domain of fibronectin. In this report, we show that peptide treatment substantially extends the survival time for mice injected intravenously with B16-F10 cells (8/8 vs. 0/8 mice alive at 150 d), thereby demonstrating the potential efficacy of GRGDS treatment in protection against metastatic colonization. We have also examined the specificity of GRGDS activity by testing a series of related homologues for their effects on experimental metastasis. The overall profile of the relative inhibitory activities of these peptides closely matched their previously established capacity to disrupt adhesion in vitro. Lung retention studies with radiolabeled B16-F10 cells revealed an accelerated rate of cell loss from the lung 0-6 h after coinjection with the active peptide GRGDS. This early effect of GRGDS was consistent with its short circulatory half-life, which was found to be 8 min. Taken together, these results suggest that peptide-mediated inhibition of pulmonary colonization is due to interference with B16-F10 cell adhesion to structures in the target organ. Possible peptide interference in tumor cell-blood cell interactions was examined in order to assess (a) possible biological side-effects of peptide treatment and (b) whether such interactions might be an alternative mechanism for GRGDS-mediated inhibition of pulmonary colonization. GRGDS was found to retain full inhibitory activity when coinjected with B16-F10 cells into mice in which platelet function was impaired by acetylsalicylic acid treatment or into thrombocytopenic mice treated with antiplatelet serum (76-93% inhibition of colony formation). These data suggest that platelet involvement in the effects of the peptide is minimal. Similarly, GRGDS was also found to be a potent inhibitor of experimental metastasis in natural killer (NK) cell-deficient beige mice (86% inhibition), thereby discounting the possibility that GRGDS artifactually enhanced NK cell activity. We conclude as a result of these studies that cell-binding fibronectin peptides are specific inhibitors of experimental metastasis that prolong survival, that they appear to function by blocking the adhesion of B16-F10 cells to structures in the target organ, and that they do not appear to act through side effects on certain metastasis-related blood cell functions. In the future, derivatives of fibronectin peptides may be potentially useful prophylactic agents for interfering with the process of metastasis.

Monoclonal antibodies specific for the neu oncogene product directly mediate anti-tumor effects in vivo.

Abstract: We have produced a panel of monoclonal antibodies which bind cell surface domains of the 185 Kd tumor antigen (p185) encoded by the neu oncogene. All of these antibodies stain neu-transformed cells in immunofluorescence assays and immunoprecipitate p185 from metabolically labeled cell lysates. All of the anti-p185 monoclonal antibodies, regardless of isotype, exert a selective cytostatic effect on the growth of neu-transformed cells suspended in soft agar, demonstrating their ability to directly inhibit the transformed phenotype. Anti-p185 antibodies of the IgM, IgG2a, and IgG2b isotypes exert a cytolytic effect on neu-transformed cells in the presence of complement. Only one IgG2a monoclonal antibody is also able to mediate minimal levels of antibody-dependent cellular cytotoxicity (ADCC) (Roussel et al., 1984) in the presence of non-immune spleen cells. In vivo administration of anti-p185 antibodies of the IgG1, IgG2a, and IgG2b isotypes exerts a profound inhibitory effect on the tumorigenic growth of neu-transformed cells. This tumor inhibitory effect is unaffected by depleting tumor bearing animals of complement, and is only minimally affected by depleting tumor bearing animals of macrophages. This suggests that neither complement-mediated killing nor ADCC are necessary for the anti-tumor effects of p185-specific monoclonal antibodies. The results presented here demonstrate that monoclonal antibodies reactive with cell surface domains of an oncogene-encoded protein can directly inhibit tumor growth in vitro and in vivo. Such antibodies may prove useful in the therapy of certain malignancies.

Evaluation of Ricin A Chain-containing Immunotoxins Directed against CD19 and CD22 Antigens on Normal and Malignant Human B-Cells as Potential Reagents for in Vivo Therapy

Abstract: Ricin A chain-containing immunotoxins (IT-As) specific for the human B-cell antigens, CD22 and CD19, were constructed using the monoclonal antibodies, HD6 and HD37, respectively. IT-As were prepared by coupling intact antibodies, F(ab')2, or Fab' fragments to native or chemically deglycosylated ricin A chain. The IT-As were then evaluated for cytotoxicity to normal and neoplastic human B-cells in vitro with the major objective of appraising their suitability for in vivo therapy of human B-cell tumors. The IT-As prepared with both the HD6 and HD37 antibodies were specifically toxic to normal B-cells and to most of the neoplastic B-cell lines tested. However, the IT-As prepared from HD6 were generally more potent than those prepared from HD37. On Daudi cells, to which the two antibodies bound in similar numbers and with similar affinities, IT-As prepared with intact HD6 antibody or its Fab' fragment were 10-fold and 1.5- to 4-fold more potent, respectively, than the corresponding HD37 IT-As. The IT-As constructed from intact HD6 antibody and native or deglycosylated A chain reduced protein synthesis in Daudi cells by 50% at a concentration of 1.2 X 10(-11) M indicating that they were only 5-fold less toxic to the cells than ricin itself. Intact HD37 IT-As produced equivalent inhibition of protein synthesis at 1.5 X 10(-10) M. With both antibodies, IT-As constructed from the Fab' fragments were 10- to 20-fold less potent than their intact antibody counterparts. Different neoplastic B-cell lines varied in sensitivity to the IT-As. In most cases, their sensitivity correlated with the levels of CD19 and CD22 antigens expressed. Neither HD6 nor HD37 IT-As affected the ability of normal human bone marrow cells to form granulocyte-macrophage colony-forming units in soft agar, suggesting that both antigens are absent from these progenitor cells. Examination of sections of frozen human tissues using immunoperoxidase staining procedures indicated that the antibodies did not bind to a panel of normal tissues lacking B-lymphocytes. These results suggest that HD6 and HD37 IT-As are candidates for in vivo therapy in humans with certain B-cell tumors. However, HD6 IT-As are more potent, reduce protein synthesis more completely, and hence appear to be the ITs of choice for treating tumors expressing the CD22 antigen.

Correlation of T and B cell activities in vitro and serum IL-2 levels in systemic lupus erythematosus.

Abstract: There have been only a few studies indicating that B cell hyperactivity in SLE could depend on Th cell activation In particular, circulating CD4+ cells were found to express Ia Our own previous investigations have shown that the decreased IL-2 secretion capacity in vitro of CD4+ cells in SLE is restored to normal when the cells are rested for a few days in culture This suggested the presence of activated, exhausted T cells in the circulation In this study, we report several observations concerning T cell function in SLE 1) Decreased IL-2 secretion in vitro of PBL was found to correlate significantly with increased spontaneous IgG secretion of such cells; immunosuppressive treatment of 22 patients with steroids plus cyclosporin A led, to a large extent, to a correction of both abnormalities 2) 9 of 18 patients with active disease (and low IL-2 secretion in vitro) had increased IL-2 levels in serum by ELISA; two sera contained IL-2 biologic activity, and chromatography of one serum showed IL-2 in a high molecular size complex (Mr approximately 50,000) dissociable with 6 M urea The serum levels of IL-2R were also frequently increased, even in less active SLE 3) In cell culture experiments, the IgG secretion by purified B cells from 6 of 9 patients with active SLE was increased by autologous T cells acting either alone (3 patients) or synergistically with rIL-2 (3 patients); the B cells from all 9 patients showed increased IL-2 responsiveness compared with blood donor B cells Taken together, these results provide new evidence that increased T cell activation occurs and plays a role in SLE

In Vitro and In Vivo Antistaphylococcal Activity of Human Stratum Corneum Lipids

Abstract: • Despite the assumption that sebum-derived fatty acids are responsible for cutaneous antimicrobial defense, no studies have assessed the contribution of epidermis-derived lipids. Herein, we tested the antistaphylococcal effects of human stratum corneum lipids, enriched in endogenous, keratinocyte-derived species obtained by lipid extraction and thin-layer chromatography, for antimicrobial activity in both in vitro and in vivo systems. Whereas the most potent species in vitro were the free fatty acids, polar lipids and glycosphingolipids also demonstrated antistaphylococcal activity in vitro, while other neutral lipids displayed virtually none, results that were confirmed with authentic standards in vitro. In a pilot study on delipidized forearm test sites in human volunteers, naturally occurring free fatty acids, polar lipids, and glycosphingolipids exhibited significantly more antistaphylococcal activity than other stratum corneum lipids or vehicle controls. Finally, biopsy specimens of incubated skin sites demonstrated penetration of staphylococci through lipid-enriched intercellular domains. These results provide the first evidence that endogenous, epidermis-derived skin lipids may contribute to cutaneous antimicrobial resistance. ( Arch Dermatol 1988;124:209-215)

In vivo immunomodulation by the neuropeptide substance P.

Abstract: Many experiments have demonstrated that the nervous and immune systems interact in a bidirectional fashion. Neuropeptides, including substance P, have been shown to modulate lymphocyte DNA, RNA and immunoglobulin synthesis in vitro and to play a role in inflammatory and hypersensitivity disease states. However, the role of substance P as an immunomodulator in vivo is uncertain and there is only indirect evidence of this effect obtained in vitro. Therefore, we have assessed the effect of substance in vivo on DNA and immunoglobulin synthesis by murine splenic and Peyer's patch lymphocytes after the continuous administration via a miniosmotic pump of substance P in vivo. Substance P administered in this fashion increased cell proliferation of lymphocytes isolated from both organs. Immunoglobulin synthesis was also increased and in a relatively isotype-specific manner. IgA synthesis was most affected, IgM synthesis less so and IgG synthesis was not changed significantly. These effects of substance P on lymphocytes in vivo are similar to its effects on cell proliferation and immunoglobulin synthesis when cells are exposed to this peptide in vitro. These results provide direct evidence that neuropeptides (substance P) may modulate lymphocyte function in vivo and that neuropeptides should be incorporated into the conceptual framework of immune regulation.

Pyrrolo[1,4]benzodiazepine antitumor antibiotics: relationship of DNA alkylation and sequence specificity to the biological activity of natural and synthetic compounds.

Abstract: The DNA alkylation and sequence specificity of a group of natural and synthetic pyrrolo-[1,4]benzodiazepines [P(1,4)Bs] were evaluated by using an exonuclease III stop assay, and the results were compared with in vitro and in vivo biological potency and antitumor activity. The P(1,4)B antibiotics are potent antitumor agents produced by various Actinomycetes, which are believed to mediate their cytotoxic effects by covalent bonding through N-2 of guanine in the minor groove of DNA. In this article we describe the results of a sensitive DNA alkylation assay using exonuclease III which permits both estimation of the extent of DNA modification as well as location of the precise guanines to which the drugs are covalently bound. Using this assay, we have evaluated a series of natural and synthetic compounds of the P(1,4)B class for their ability to bind to DNA and also determined their DNA sequence preference. The compounds included in this study are P(1,4)Bs carrying different substituents in the aromatic ring, having varying degrees of saturation in the five-membered ring, or differing in the stereochemistry at C-11a. These same compounds were evaluated for in vitro cytotoxic activity against B16 melanoma cells, for potency in vivo in B6D2F1 mice (LD50), and for antitumor activity (ILSmax) against P388 leukemia cells. A good correlation was found between extent of DNA alkylation and in vitro and in vivo potency. Furthermore, on the basis of electronic and steric considerations, it was possible to rationalize why those compounds that showed negligible biological activity were unable to bond covalently to DNA. Last, we have determined that the degree of saturation in the five-membered ring of the P(1,4)Bs has a significant effect on the DNA bonding reactivity and biological activity of this class of compounds.

Production of peptides inducing chemotaxis and lysosomal enzyme release in human neutrophils by intestinal bacteria in vitro and in vivo.

Abstract: Chadwick VS, Mellor DM, Myers DB, Selden AC, Keshavarzian A, Broom MF, Hobson CH. Production of peptides inducing chemotaxis and lysosomal enzyme release in human neutrophils by intestinal bacteria in vitro and vivo.Low molecular weight (MI 200-1500) N-formylated peptides that stimulate many leucocyte functions, including chemotaxis and lysosomal enzyme release, have previously been isolated from Escherichia coli cultures. We have used high-performance liquid chromatography and bioassay techniques to study production of such peptides by intestinal bacteria in vitro and their activity in intestinal luminal contents, obtained by in vivo dialysis methods. Bioactivity was detected in culture supernatants of all 11 species of bacteria so far investigated, was resistant to digestion with amino-peptidase, but was destroyed by carboxypeptidase, confirming that bioactive moieties were amino-terminal-blocked peptides. By similar isolation procedures, pronase-sensitive bioactive factors have been demonstrated in hum...

A mycoplasma high-affinity transport system and the in vitro invasiveness of mouse sarcoma cells.

Abstract: FS9 mouse sarcoma cells were previously shown to be highly invasive when confronted with chicken heart fibroblasts using Abercrombie's confronted explant technique. This invasion could be inhibited by addition to the assay of Fab fragments of a monoclonal antibody directed against p37, a protein associated with the surface of FS9 cells. We have cloned and sequenced the gene for p37. We show that it originates from Mycoplasma hyorhinis and that UGA is a tryptophan codon in this organism. We present evidence that the p37 gene is part of an operon encoding two additional proteins which are highly similar to components of the periplasmic binding-protein-dependent transport systems of Gram-negative bacteria, and we suggest that p37 is part of a homologous, high-affinity transport system in M. hyorhinis, a Gram-positive bacterium. We discuss the influence of p37 and M. hyorhinis on contact inhibition of locomotion of mammalian cells.

Dimerization of the tat protein from human immunodeficiency virus: a cysteine-rich peptide mimics the normal metal-linked dimer interface.

Abstract: We have synthesized an 18-amino acid peptide that contains the cysteine-rich region of the tat protein from human immunodeficiency virus. Previous experiments in vitro with the intact tat protein have shown that these cysteines serve as metal ligands, causing tat to form metal-linked dimers. Ultraviolet absorption spectra show that the synthetic peptide (tat21-38) binds two Cd2+ or two Zn2+ ions per peptide monomer, and some changes in the circular dichroism spectra are seen as the metals bind. The peptide-metal complexes are completely resistant to proteolytic digestion, and mass spectrometry demonstrates that this peptide forms metal-linked dimers. The peptide can also combine with the intact tat protein to form metal-linked heterodimers. If these heterodimers are unable to trans-activate viral transcription, tat21-38 could be a lead compound for designing drugs to treat acquired immunodeficiency syndrome.

Active suppression of host-vs graft reaction in pregnant mice. IX. Soluble suppressor activity obtained from allopregnant mouse decidua that blocks the cytolytic effector response to IL-2 is related to transforming growth factor-beta.

Abstract: Non-Ts cells in murine allopregnancy decidua release potent immunosuppressor factors in vitro that block the action of IL-2. Previous studies have shown that both primary and secondary CTL responses are inhibited as well as the generation of Il-2 activated killer cells. In this paper we show that the suppressor factor(s) can arrest ongoing IL-2 dependent CTL responses but does not block binding of anti-IL-2R antibody or radiolabeled IL-2 to the IL-2R. The suppressive activity is associated with molecules that adhere to hydroxylapatite and con A-agarose but do not bind to activated charcoal or partition as lipids. HPLC TSK 3000 separation showed a major peak of suppressive activity at 60 to 100 kDa, with additional activity at 300 kDa, and at less than 1000. Under acid conditions, suppressive activity resolved as a major peak at 13 kDa with some residual activity at 65 kDa and at less than or equal to 1000. A specific rabbit IgG antibody to transforming growth factor-beta neutralized suppressor activity in unseparated supernatant and in the 13-kDa fraction whereas neutralizing antibodies to progesterone or PGE-2 did not affect suppression but could neutralize their respective ligands. Inasmuch as transforming growth factor-beta has a 25 kDa Mr, the 13-kDa decidua-associated suppressor factor would appear to represent a related but distinct regulatory molecule that associates with a variety of carrier molecules.

In vitro transcription and translation of bluetongue virus mRNA.

Abstract: Fractionation of in vitro transcribed bluetongue virus (BTV) mRNA by agarose gel electrophoresis resulted in the separation of eight of the 10 species. The relative molar ratio of the mRNAs confirmed that mRNA 5 was transcribed more frequently than would be predicted from the size of the S5 genome segment, while mRNA 10 was transcribed less frequently. In vitro translation of unfractionated BTV mRNAs resulted in the synthesis of the seven known structural proteins (P1 to P7) and two known non-structural proteins (NS1 and NS2). Two additional non-structural proteins (NS3 and NS3A) with Mr of 28K and 25K respectively were identified. The protein coding assignments for the medium- and small-sized double-stranded RNA genome segments of BTV serotype 10 were found to correspond to those reported for BTV-1 and BTV-17. The peptide maps of NS1, NS2, NS3 and NS3A synthesized in vitro corresponded to those of their counterparts synthesized in infected cells. Protein NS3A appeared to be a truncated form of NS3, since its peptide map completely overlapped that of NS3. Proteins NS3 and NS3A were present in very small amounts in the soluble fraction of the cytoplasm of infected cells, and were synthesized in variable amounts in vitro, whereas the other nine viral proteins were synthesized in constant molar ratios. A difference in the relative molar ratios in which some of the BTV proteins were synthesized in vitro and in vivo was observed. In vivo, protein NS1 was translated in the largest amount but in vitro, NS2 was the most efficiently translated protein. Conversely, protein P6 was translated much more efficiently in vitro than in vivo.

Effects of a grooved epoxy substratum on epithelial cell behavior in vitro and in vivo

Abstract: The effects of grooved epoxy substrata on epithelial (E) cell behavior were studied in vitro and in vivo. V-shaped grooves, 10 microns deep, were produced in silicon wafers by micromachining, a process which was developed for the fabrication of microelectronic components. The grooved substrata were replicated in epoxy resin. More E cells attached to grooved surfaces than to adjacent smooth surfaces. Clusters of E cells were markedly oriented by the grooved surfaces in comparison to the adjacent smooth surfaces where the orientation was random. Grooved and smooth epoxy implants were placed percutaneously in the parietal area of rats. One week after implantation E cells were found to adhere tightly to the implant surfaces. In the grooved portion of the implant E cells interdigitated into the grooves and had rounded nuclei. Histomorphometric measurements indicated that there was a shorter length of epithelial attachment and a longer length of connective tissue attachment in the grooved, compared to the smooth, portion of implants. After 10 days the epithelial attachment had migrated down the length of the protruding smooth portion of the implant and was located on the base of the implant. However, epithelium remained attached to the grooved portion of the implant. These observations indicate that grooved surfaces have the potential to impede epithelial downgrowth on percutaneous devices.

Human recombinant IL-6/B cell stimulatory factor 2 augments murine antigen-specific antibody responses in vitro and in vivo.

Abstract: The effects of human rIL-6/B cell stimulatory factor 2 (hrIL-6/BSF-2) from Escherichia coli on murine Ag, SRBC-specific antibody responses were examined in vitro and in vivo. HrBSF-2 was effective in augmenting the primary and the anamnestic plaque-forming cells response to SRBC in vitro. The augmentation of the primary response was apparent when B cell-enriched spleen cells (B cells) were cultured with BSF-2 in the presence of IL-2. On the other hand, hrBSF-2 alone strongly enhanced the anamnestic response in a dose-dependent manner when spleen cells from SRBC-immunized mice were used. These effects of BSF-2 were abolished completely by anti-BSF-2 antibody, but not by normal rabbit Ig. Cell depletion experiments indicated that L3T4 (CD4)+ T cells, but not Lyt-2(CD8)+ T cells, and adherent cells (macrophages) have an important role in this BSF-2-induced augmentation of the response. In addition, kinetic studies showed that hrBSF-2 acts on B cells in the anamnestic response even when added relatively late in the culture. Finally, it was determined whether BSF-2 also could be active in modulating antibody responses in vivo. BSF-2 was shown to enhance the primary and secondary antibody responses in mice. The most apparent effect of BSF-2 was observed in the secondary response.

Modulation of Murine Tumor Major Histocompatibility Antigens by Cytokines in Vivo and in Vitro

Abstract: The capacity of different cytokines to upregulate major histocompatibility complex (MHC) expression on murine tumor cells in vitro, and on s.c. tumors or pulmonary metastases in vivo has been examined. Interleukins-1, -2, and -4 (IL-1, -2, -4), tumor necrosis factor-alpha (TNF-alpha), alpha-interferon (IFN-alpha), and gamma-interferon (IFN-gamma), were incubated with tissue culture lines of murine tumor cells displaying low (MCA-101), intermediate (MCA-102, -106), or high (MCA-105) Class I expression. IFN-alpha and IFN-gamma significantly increased Class I but not Class II antigens on all lines. TNF-alpha, IL-1, -2, and -4 had no significant effect on Class I or II expression in vitro. Mice bearing pulmonary metastases or s.c. lesions generated by MCA-101 and -102 were treated with IFN-alpha or IFN-gamma i.p. or i.v., or with a single dose of TNF-alpha i.v. Immunoperoxidase staining of lung metastases or subcutaneous tumors showed an increase in Class I but not Class II expression on MCA-102 tumors treated with IFN-alpha or IFN-gamma. IL-1, -2, or TNF-alpha had no effect on MHC Class I or II expression in vivo. None of the cytokines tested could upregulate MHC Class I or II expression on MCA-101 tumors in vivo. Flow cytometry analysis demonstrated an increase in Class I but not Class II expression on MCA-102 and MCA-106 tumor cells from s.c. tumor treated with IFN-alpha or IFN-gamma. A kinetic analysis of the flow cytometry data revealed that augmented MCA-102 Class I levels persisted for several days after cessation of in vivo therapy with IFN-alpha. Our data suggest one possible mechanism for the synergistic antitumor effects of IL-2 and IFN-alpha.

IgE-BINDING FACTORS AND REGULATION OF THE IgE ANTIBODY RESPONSE

Abstract: In rodents, IgE-bF are derived from a subset of T cells that bear Fc epsilon R or Fc gamma R, or both, and selectively enhance or suppress the IgE response. IgE-PF and IgE-SF may share a common structural gene, therefore a common polypeptide chain, and their biologic activities are decided by post-translational glycosylation process. Under physiological conditions, this process is controlled by two lymphokines, i.e. GEF and GIF. The same principle probably applies to human T cell-derived IgE-bF. In both rodent and human lymphocytes, Fc epsilon RII on B cells are degraded, and their fragments are released from the cells. The fragments of Fc epsilon RII on human B cells represent the carboxy terminal half of the receptor molecules and have affinity for IgE. In contrast, the fragment of Fc epsilon R in mouse B cells does not have an affinity for IgE. Thus, "IgE-bF" are derived from both T cells and B cells in humans, but only from T cells in rodents. The formation of T cell-derived IgE-bF was induced by interferons, while biosynthesis of Fc epsilon R in B cells and the formation of their fragments were enhanced by IL-4. IgE-bF are also formed by a subset of antigen-primed T cells upon cognate interaction with antigen-pulsed syngeneic macrophages. These antigen-primed T cells constitutively secrete either GEF or GIF, having no affinity for homologous antigen. Upon antigenic stimulation, however, GEF and GIF formed by the cells had affinity for the antigen. The antigen-specific GEF enhanced the antibody response, and antigen-specific GIF suppressed the antibody response, both in carrier specific manner. The possible relationship between antigen-specific GEF and antigen-specific TaF, and that between antigen-specific GIF and antigen-specific TsF both require further studies. Nonspecific GIF not only switches T cells from the formation of IgE-PF to the formation of IgE-SF, it also facilitates the generation of antigen-specific suppressor T cells which produce antigen-specific GIF upon antigenic stimulation. Propagation of antigen-primed T cells in the presence of GIF also facilitate the generation of antigen-specific suppressor T cells in vitro. If the same procedures would be effective for human T cells of allergic patients, it would be possible to generate antigen-specific suppressor T cells from their T cell population in vitro and to establish T cell hybridomas that produce allergen-specific GIF(TsF).(ABSTRACT TRUNCATED AT 400 WORDS)