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L A Falk

Publications and source records attributed to L A Falk.

At least 19 recordsLinked to original sources
PubMed2001

Prelicensure evaluation of combination vaccines.

There is considerable public health interest in licensing safe and effective combination vaccines. Because combination vaccines may progress rapidly from phase 1 to a pivotal phase 2 immunogenicity trial, a rigorous approach to address product issues early in development is warranted. Clinical studies to evaluate the safety, immunogenicity, and (when necessary) clinical end point efficacy of combination vaccines should be randomized and well controlled in most cases. A large phase 3 safety study (i.e., a study that enrolls thousands of vaccinees) should be included in the development plan if a phase 3 (clinical end point) efficacy trial will not be conducted. Often, the new combination vaccine under development contains immunogens that have all been previously licensed, have demonstrated efficacy in earlier clinical trials, or both. For such products, comparative immunogenicity data may be sufficient to support efficacy. When applicable, clinical data to support simultaneous administration with other relevant vaccines should be obtained. Given the complexity of combination vaccine development, early consultation with United States Food and Drug Administration can be invaluable.

Clinical Trials as Topic↗
PubMed2001

Evaluating the immune response to combination vaccines.

Assessment of the immune responses to combination vaccines in the United States has generally been based on randomized, controlled comparative trials, with such studies designed to rule out predefined differences. In designing clinical studies of the immune response to combination products, attention should be directed toward selecting the appropriate immunologic end points and control groups. Acceptable differences in immune responses between combination and control groups should be predefined, and an adequate statistical plan should be developed. In many cases, it may be necessary to evaluate simultaneous administration of other recommended vaccines, assess schedule changes for 1 or more components of a combination, and bridge immunologic data obtained from international studies to the population of the United States. We discuss the use of immunogenicity studies to support the licensure of combination vaccines when field efficacy studies are either not possible or not required and highlight some recent experiences with combination vaccines containing Haemophilus influenzae type b polysaccharide conjugates.

Antibodies, Bacterial↗
PubMed2001

Manufacturing issues with combining different antigens: a regulatory perspective.

The regulation of biological products is conducted within the framework Title 21 of the US Code of Federal Regulations (CFR). These regulations describe product and clinical testing requirements for drugs and biological products, as well as the requirements for licensure of such products. The requirements outlined in the CFR also apply to combination vaccines. In addition, the Center for Biologics Evaluation and Research has issued a Guidance to Industry document that discusses the manufacturing, testing, and clinical evaluation of combination vaccines. However, as the complexity of mixing the different antigens increases, the challenges associated with product development (e.g., demonstration of comparability of the components and lot consistency) require early interactions with the US Food and Drug Administration. The many areas of difficulty in the arena of combination vaccine development underscore the need for continued reevaluation of current guidance documents in addressing the increasing complexity of vaccines.

Adjuvants, Immunologic↗
PubMed2001

Current status and future trends in vaccine regulation--USA.

In regulating vaccines, the US Food and Drug Administration (FDA) is governed by the Code of Federal Regulations. These regulations serve as the framework for product characterization, as well as preclinical and clinical testing strategies. Novel vaccine approaches such as combination vaccines, vectored vaccines, new adjuvants, and novel delivery systems pose unique regulatory challenges for the FDA. If US licensure is sought, communication with the FDA throughout the clinical development of a product is essential to identify and implement the appropriate strategies for demonstrating the safety and effectiveness of a new product.

Adjuvants, Immunologic↗
PubMed2000

Recombinant bacillus Calmette-Guérin as a potential vector for preventive HIV type 1 vaccines.

In August 1997, the World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS) convened an expert working group to discuss current strategies for the development of HIV type 1 vaccines. Based on the recent findings of investigators from Japan's National Institute of Infectious Diseases (NIID) in Tokyo using recombinant bacillus Calmette-Guérin (rBCG) as a potential vectored vaccine for HIV, a recommendation was made that further work in this area is a priority. As a result, the working group reconvened in September 1998 to discuss the progress to date with this vaccine approach, as well as areas of related research to assess the feasibility of a BCG-vectored HIV vaccine. This report summarizes the discussions addressing the available scientific data on the potential use of rBCG as a vector for preventive HIV vaccines, the work necessary to move such candidate vaccines into Phase 1 clinical trials, and recommendations targeted at facilitating the long-term development of rBCG-vectored HIV vaccines.

AIDS Vaccines↗
PubMed1998

Review of current preclinical testing strategies for bacterial vaccines.

A wide variety of bacterial vaccines is in various stages of preclinical and clinical development. These products range from whole killed or live attenuated bacterial organisms to purified proteins, peptides and plasmid DNA. Although preclinical strategies may be directed by a set of common guidelines focused on demonstrating safety and biological activity, the exact developmental scheme will depend on product-specific characteristics. In general, preclinical data should support the proposed clinical formulation and include detailed information on the source and quality of starting materials, manufacturing processes, characterization of bacterial seed stocks, potency, general safety, purity, and identity. Data describing product validation and testing may be appropriate depending on the type of product, e.g., genetic stability for recombinant constructs, details on inactivation or attenuation methods for organisms or toxins, demonstration of potency of combination products, and safety and toxicology studies of plasmid DNA vaccines or vaccines with novel adjuvants. The choice of dose, route, and formulation to be used clinically may be greatly affected by rigorous preclinical developmental strategies.

Adjuvants, Immunologic↗
PubMed1997

Colchicine down-regulates lipopolysaccharide-induced granulocyte-macrophage colony-stimulating factor production in murine macrophages.

Activation of macrophages by LPS and taxol results in production of IL-1, IL-6, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF), which are involved in regulating hemopoiesis, inflammation, and immune responses. Microtubules are proposed as a target site for LPS interaction(s), based on similarities between the effects of the tubulin-binding drug taxol and LPS. To clarify the role of microtubules in LPS-induced GM-CSF expression in macrophages, we examined whether microtubule depolymerizing agents affect GM-CSF production in macrophages. Pretreatment with colchicine impaired LPS induction of GM-CSF in RAW 264 cells, and studies using stable transfectants revealed that colchicine impaired the transcriptional responsiveness of a reporter gene driven by a GM-CSF promoter sequence. Colchicine inhibition of the GM-CSF response correlated with decreases in the mRNA levels of beta-tubulin; maximal inhibition of both events was observed 4 h after addition of colchicine. Microtubule agents inhibited LPS induction of IL-6 and TNF-alpha, while the induction of both IL-1beta and inducible nitric oxide synthase was unaltered, suggesting that LPS activates microtubule-dependent and -independent pathways. Interestingly, LPS stimulation of macrophages down-regulated levels of beta-tubulin transcripts, implying that LPS interacts with an element(s) of the microtubule network in vivo, activating pathways regulating transcription of beta-tubulin. The ability of both colchicine and LPS to modulate transcription of beta-tubulin suggests that this event does not per se underlie the inhibitory effect of colchicine on LPS-induced GM-CSF expression. These data led us to conclude that colchicine inhibits LPS induction of GM-CSF by affecting microtubule-dependent costimulatory signaling pathways that synergize with primary LPS-triggered responses.

Animals↗
PubMed1997

IL-6 induction of protein-DNA complexes via a novel regulatory region of the inducible nitric oxide synthase gene promoter: role of octamer binding proteins.

Macrophage inducible nitric oxide synthase (iNOS) catalyzes the synthesis of NO. IL-6-stimulated macrophage differentiation of murine myeloid M1 cells is accompanied by iNOS gene induction and steady-state mRNA expression. Two regions within the iNOS promoter mediate transcriptional responsiveness to LPS and IFN-gamma. Region I contains several essential transcription factor binding motifs and promotes responsiveness to LPS, whereas region II potentiates the LPS response by IFN-gamma. Because region I possesses basal promoter activity and directly mediates iNOS gene activation, we attempted to identify the trans-acting factors involved in IL-6-stimulated induction of the murine iNOS gene through this region. Using an electrophoretic mobility shift assay and methylation interference, we show that IL-6 induced reciprocal changes in the binding activity of POU family members to the candidate nonconsensus octamer sequence of region I that correlated, temporally, with iNOS steady-state mRNA expression. Although DNA-protein binding activity of IL-6-stimulated whole-cell extracts also interacted with a radiolabeled canonical octamer motif, such DNA-protein complexes were not eliminated in competition assays using consensus nuclear factor kappaB or IL-6 oligonucleotides. Specifically, our studies show that octamer binding protein-1-related protein binding activity decreased, while binding of octamer binding protein-2-related proteins increased during differentiation. Mutation of the octamer motif disrupted both binding of the IL-6-induced protein-DNA interactions and transcriptional activation through region I, revealing that this motif is absolutely essential for IL-6 induction of iNOS. Thus, differential activation of octamer binding transcriptional modulators from the POU family may be a novel mechanism of IL-6-mediated iNOS gene regulation.

Animals↗
PubMed1996

Transforming growth factor beta 1 functions in monocytic differentiation of hematopoietic cells through autocrine and paracrine mechanisms.

This study examined the role of transforming growth factor beta 1 (TGF-beta 1) in monocytic differentiation of hematopoietic cells. TGF-beta 1 and retinoic acid (RA) inhibited HL-60 cell growth in a dose-dependent fashion. Treatment of HL-60 cells with a combination of TGF-beta 1 and a 50% optimal dose of RA (RA + TGF-beta 1) resulted in increased growth suppression compared to the individual treatments. Morphological studies revealed that TGF-beta 1 induced promonocytic differentiation (68%), RA induced granulocytic differentiation (98%), and RA + TGF-beta 1 induced monocytic (54%) and granulocytic (46%) differentiation of HL-60 cells. Induction of the monocyte-specific marker, nonspecific esterase, was markedly increased by TGF-beta 1 and RA + TGF-beta 1 treatment but not by RA treatment. Both TGF-beta 1 treatment and RA treatment increased TGF-beta ligand and TGF-beta receptor protein and mRNA levels. To determine whether RA mediated HL-60 cell growth inhibition and differentiation through the autocrine expression of TGF-beta 1, experiments using TGF-beta 1 antisense oligonucleotides or TGF-beta 1-neutralizing antibodies were conducted. TGF-beta 1 antisense oligonucleotides and neutralizing antibodies partially blocked RA-induced inhibition of proliferation, and TGF-beta 1 antisense oligonucleotides reversed RA-induced granulocytic maturation, demonstrating that RA signals autocrine expression of TGF-beta 1 and TGF-beta receptors. The effect of TGF-beta 1 on normal hematopoiesis was also studied using primary human fetal liver cells. TGF-beta 1 alone and in the presence of interleukin 3 promoted macrophage differentiation of primitive fetal liver cells. Cell surface expression of the monocyte/macrophage-specific marker c-fms was increased 3.1-fold following TGF-beta 1 treatment. In addition, TGF-beta 1-treated cells displayed a 51% increase in phagocytosis as compared to interleukin 3-treated control cells. These studies define a role for TGF-beta 1 in the autocrine and paracrine regulation of monocyte/macrophage differentiation.

Biomarkers↗
PubMed1995

Human interleukin-3 receptor modulates bcl-2 mRNA and protein levels through protein kinase C in TF-1 cells.

Upon withdrawal of interleukin-3 (IL-3) from human factor-dependent erythroleukemic cell line TF-1, bcl-2 mRNA and protein levels decrease within 8 to 24 hours. Accompanying this decrease is the onset of apoptosis as determined by flow cytometric analysis of DNA degradation. By 8 to 18 hours of deprivation approximately 70% to 80% of the cells have entered apoptosis. Downregulation of protein kinase (PK) by a 24-hour incubation in 100 nmol/L 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the presence of IL-3 dramatically reduced bcl-2 mRNA levels, and induced apoptosis in the presence of IL-3. We have also found that even in the presence of IL-3, two inhibitors of PKC, light-activated calphostin and H-7, substantially reduced the levels of bcl-2 mRNA between 8 and 24 hours as measured by a semi-quantitative reverse transcriptase/polymerase chain reaction assay method; however, the cyclic nucleotide-dependent PK inhibitor HA 1004, that is a structural analog of H-7 but a poor inhibitor of PKC, did not reduce bcl-2 levels in the presence of IL-3. This decrease in bcl-2 mRNA was accompanied by a decline in bcl-2 protein levels by 8 to 24 hours after addition of light-activated calphostin. In addition to interfering with the maintenance of bcl-2 mRNA levels, inhibition of PKC with H-7 inhibited the induction of bcl-2 mRNA in factor-deprived TF-1 cells restimulated with IL-3. The cyclic nucleotide-dependent PK inhibitor HA 1004 did not inhibit IL-3-induced bcl-2 mRNA. Studies with actinomycin D showed that transcription plays a major role in maintaining bcl-2 levels in TF-1 cells, and it is therefore likely that IL-3 plays a role in maintaining bcl-2 transcription through activation of PKC in these cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Bidirectional effects of transforming growth factor beta (TGF-beta) on colony-stimulating factor-induced human myelopoiesis in vitro: differential effects of distinct TGF-beta isoforms.

Transforming growth factor-beta (TGF-beta) has potent antiproliferative effects on human hematopoietic progenitor cells. We report here that TGF-beta 1 and -beta 2 also exert bimodal dose-dependent stimulation of granulocyte-macrophage colony-stimulating factor (CSF) and granulocyte-CSF-induced day 7 granulocyte-macrophage colony-forming units. This increase in colony formation was restricted to low doses (0.01 to 1.0 ng/mL) of TGF-beta 1 and was due to increased granulopoiesis, showing that TGF-beta can affect the differentiation as well as the proliferation of hematopoietic progenitors. Furthermore, TGF-beta 3 was found to be a more potent inhibitor of hematopoietic progenitor cells than TGF-beta 1 and -beta 2. In contrast to the bidirectional proliferative effects of TGF-beta 1 and -beta 2, the effects of TGF-beta 3 on human hematopoiesis were only inhibitory, showing for the first time that TGF-beta isoforms differ not only in potencies but also with regard to the nature of the response they elicit.

Cell Differentiation↗
PubMed1991

Mechanistic studies of transforming growth factor-beta inhibition of IL-2-dependent activation of CD3- large granular lymphocyte functions. Regulation of IL-2R beta (p75) signal transduction.

CD3- large granular lymphocyte (LGL) express constitutive levels of functional IL-2R beta. TGF-beta inhibited several IL-2R beta-mediated events in LGL, including IL-2-induced NK and lymphokine-activating factor activities, IFN-gamma gene expression and secretion, and IL-2R alpha expression. TGF-beta inhibited these IL-2-induced LGL functions in a dose-dependent and reversible manner. By contrast, TGF-beta had little effect on LGL IL-2R beta expression and TGF-beta receptors were not induced by IL-2. Studies were performed to examine binding and internalization of radiolabeled IL-2. These experiments demonstrated that the rapid binding and internalization of [125I]IL-2 was not altered in CD3- LGL pretreated with TGF-beta. These internalization studies indicated that the TGF-beta inhibition represented postreceptor-binding events in NK cells. Further studies were initiated to examine signaling events in CD3- LGL. When IL-2-induced tyrosine phosphorylation events were examined, significant inhibition was seen of selected phosphoproteins in TGF-beta-pretreated cells. In addition, the ability of TGF-beta to also inhibit IL-2 induction of LGL IL-2R alpha and IFN-gamma mRNA expression was consistent with the hypothesis that posttranscriptional mechanisms were unlikely to be affected by TGF-beta. Collectively, these data indicated that TGF-beta inhibited IL-2-induced CD3- LGL functions and suggested that TGF-beta inhibition occurs either at the level of specific tyrosine phosphorylation and/or IL-2-induced transcriptional control factors.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Differential expression of transforming growth factor-beta 1 (TGF-beta 1) receptors in murine myeloid cell lines transformed with oncogenes. Correlation with differential growth inhibition by TGF-beta 1.

Transforming growth factor-beta 1 (TGF-beta 1) is a pleiotropic polypeptide hormone known to play an important role as a modulator of hematopoietic processes in human and murine cells. One of the characteristics of TGF-beta 1 is the ability to inhibit the growth of several cell types, including cells of the myeloid lineage. To study the mechanism by which TGF-beta 1 inhibits the growth of myeloid cells, we have used three murine myeloid cell lines, the parental interleukin-3-dependent 32D-123 cell line and two retrovirally infected interleukin-3-independent cell lines (32D-abl, 32D-src), all of which are growth inhibited by TGF-beta 1. Each of these oncogene-transfected cells expresses a greater number of TGF-beta 1 receptors than the parental cell line and responds to TGF-beta 1 with increased sensitivity; 32D and 32D-src cells are 2- and 58-fold more sensitive to TGF-beta 1 inhibition than the parental cell line (ED50 = 35 pM). Both 32D-abl- and 32D-src-transformed cell lines expressed higher levels of the 65- and 85-kDa TGF-beta 1 receptor species than did the parental cells. We observed a correlation between the greater sensitivity of 32D-src cells to TGF-beta 1 and the more rapid down-modulation and reappearance of cell surface TGF-beta 1 receptors on 32D-src cells. Thus, the level of TGF-beta 1 receptor expression and rate of reexpression both have a crucial regulatory effect on the functional activity of the TGF-beta 1 ligand.

Animals↗
PubMed1991

Induction of transforming growth factor-beta 1 (TGF-beta 1), receptor expression and TGF-beta 1 protein production in retinoic acid-treated HL-60 cells: possible TGF-beta 1-mediated autocrine inhibition.

Treatment of HL-60 cells, a human promyelocytic leukemia cell line, with the vitamin A derivative retinoic acid (RA) for 7 days resulted in a dose-dependent decrease in proliferation and increase in granulocytic differentiation. The role of transforming growth factor-beta 1 (TGF-beta 1), a protein with pleiotropic effects on the proliferation and differentiation of various cell types, was examined during RA-induced differentiation of HL-60 cells. Although TGF-beta 1 alone had little effect on proliferation or differentiation of HL-60 cells, addition of TGF-beta 1 to HL-60 cells treated with a suboptimum concentration of RA (1.0 nmol/L) resulted in a marked decrease in proliferation with no effect on granulocytic differentiation. Studies of the mechanism of RA-induced TGF-beta sensitivity showed that although untreated HL-60 cells expressed low levels of TGF-beta 1 binding proteins on the cell surface, the levels were increased in a dose-dependent manner after RA treatment. Maximum induction was achieved after treatment with 10 nmol/L RA and consisted predominantly of the 65-Kd TGF-beta 1 receptor type. Moreover, RA treatment also resulted in a dose-dependent increase in both TGF-beta 1 steady-state mRNA expression and production of active TGF-beta with maximum induction at 10 nmol/LRA. RA treatment of HL-60 cells had no effect on TGF-beta 2 and TGF-beta 3 mRNA expression. These data suggest that the effects of RA may be mediated by a TGF-beta 1-mediated autocrine antiproliferative loop during differentiation of HL-60 cells.

Cell Differentiation↗
PubMed1991

In vivo and in vitro effects of TGF-beta 1 on normal and neoplastic haemopoiesis.

TGF-beta 1 and TGF-beta 2 are equipotent selective inhibitors of murine and human haemopoiesis in vitro. Primitive haemopoietic cells such as the high proliferative potential progenitor cell and the colony-forming unit (CFU)-GEMM are directly inhibited by TGF-beta whereas the more differentiated CFU-G, CFU-M and CFU-E are not. Recombinant TGF-beta 1 administered intraperitoneally or intravenously to mice selectively inhibits haemopoietic colony formation in a time- and dose-dependent manner to the same extent as seen in vitro. The progenitors are reversibly prevented from entering the cell cycle. This inhibitory action of TGF-beta functions on at least two levels: (1) down-modulation of the cell surface expression of receptors for growth stimulatory molecules and (2) interference with the intracellular signalling pathways of these molecules. In addition, expression of TGF-beta receptors is regulated during cytokine stimulation of haemopoiesis. Neoplastic B lymphocytes can proliferate by escaping from a TGF-beta-mediated autocrine inhibitory loop. Activation signals (e.g. phorbol esters) inhibit tumour cell growth by stimulating active TGF-beta production and inducing cell surface expression of TGF-beta receptors. These results indicate that TGF-beta may be useful as a bone marrow protective and/or an antitumour agent.

Animals↗
PubMed1990

Inhibition of murine monocyte proliferation by a colony-stimulating factor-1 antisense oligodeoxynucleotide. Evidence for autocrine regulation.

Using a combination of v-myc and v-ras oncogenes, we have established a growth factor-independent monocyte cell line from murine fetal liver (FL-ras/myc). Biologic and molecular characterization demonstrated that the gene for the macrophage growth factor CSF-1 and the c-fms proto-oncogene (CSF-1 receptor) are expressed in this cell line, thus suggesting autocrine regulation as a possible mechanism for the unregulated growth of these cells. To study this possibility, we used 1) mAb, to neutralize the CSF-1 protein produced by the cell line, and 2) antisense oligomers, to inhibit CSF-1 gene products by specific base-pairing of complementary nucleic acids. We report here that both approaches inhibited in vitro cell growth by 60 to 70%, whereas the combination of oligomer and mAb inhibited proliferation by 95%. However, control antisense oligomers (50% bp mismatch with CSF-1 mRNA) did not inhibit FL-ras/myc cell growth. Furthermore, the inhibitory effects of mAb and oligomers were reversible when they were removed from the media. Detection of cell-associated CSF-1 protein by immunofluorescence showed that cells treated with the antisense oligomer expressed significantly less CSF-1 protein. These results indicate that the FL-ras/myc cell line requires CSF-1 for autonomous growth and that oligomers can efficiently block production of autocrine growth factors.

Animals↗
PubMed1990

Transforming growth factor beta is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action.

Transforming growth factor beta (TGF-beta) acts as a potent inhibitor of the growth and functions of lymphoid and hemopoietic progenitor cells. Cell proliferation depends not only on the presence of growth factors, but also on the development of specific receptor-signal transducing complexes. We therefore investigated whether the inhibitory actions of TGF-beta could be mediated by inhibition of growth factor receptors. TGF-beta inhibited the constitutive level of interleukin 1 receptor (IL-1R) expression on several murine lymphoid and myeloid progenitor cell lines, as well as IL-1R expression induced by interleukin 3 (IL-3) on normal murine and human bone marrow cells. Furthermore, treatment of bone marrow progenitor cells with TGF-beta concomitantly inhibited the ability of IL-1 to promote high proliferative potential (HPP) colony formation as well as blocked IL-1-induced IL-2 production by EL-4 6.1 cells. These findings provide the first evidence that the inhibitory action of TGF-beta on the growth and functional activities of hematopoietic and T cells is associated with a reduction in the cell surface receptor expression for IL-1.

Animals↗