Biomedical subjects

D M Rothstein

Publications and source records attributed to D M Rothstein.

At least 19 recordsLinked to original sources

PubMed2001

CD45 is a JAK phosphatase and negatively regulates cytokine receptor signalling.

The regulation of tyrosine phosphorylation and associated signalling through antigen, growth-factor and cytokine receptors is mediated by the reciprocal activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPases). The transmembrane PTPase CD45 is a key regulator of antigen receptor signalling in T and B cells. Src-family kinases have been identified as primary molecular targets for CD45 (ref. 4). However, CD45 is highly expressed in all haematopoietic lineages at all stages of development, indicating that CD45 could regulate other cell types and might act on additional substrates. Here we show that CD45 suppresses JAK (Janus kinase) kinases and negatively regulates cytokine receptor signalling. Targeted disruption of the cd45 gene leads to enhanced cytokine and interferon-receptor-mediated activation of JAKs and STAT (signal transducer and activators of transcription) proteins. In vitro, CD45 directly dephosphorylates and binds to JAKs. Functionally, CD45 negatively regulates interleukin-3-mediated cellular proliferation, erythropoietin-dependent haematopoieisis and antiviral responses in vitro and in vivo. Our data identify an unexpected and novel function for CD45 as a haematopoietic JAK phosphatase that negatively regulates cytokine receptor signalling.

Animals↗

PubMed2001

Targeting signal 1 through CD45RB synergizes with CD40 ligand blockade and promotes long term engraftment and tolerance in stringent transplant models.

The induction and maintenance of allograft tolerance is a daunting challenge. Although combined blockade of CD28 and CD40 ligand (CD40L)-costimulatory pathways prevents allograft rejection in some murine models, this strategy is unable to sustain engraftment in the most immunogenic allograft and strain combinations. By targeting T cell activation signals 1 and 2 with the novel combination of anti-CD45RB and anti-CD40L, we now demonstrate potent enhancement of engraftment in C57BL/6 recipients that are relatively resistant to costimulatory blockade. This combination significantly augments the induction of tolerance to islet allografts and dramatically prolongs primary skin allograft survival. Compared with either agent alone, anti-CD45RB plus anti-CD40L inhibits periislet infiltration by CD8 cells, B cells, and monocytes; inhibits Th1 cytokines; and increases Th2 cytokine expression within the graft. These data indicate that interference with activation signals one and two may provide synergy essential for prolonged engraftment in situations where costimulatory blockade is only partially effective.

Adjuvants, Immunologic↗

PubMed2001

CTLA-4 up-regulation plays a role in tolerance mediated by CD45.

Cytolytic T lymphocyte-associated antigen 4 (CTLA-4) is a critical down-regulatory molecule in T cells that plays a major role in peripheral tolerance. Although the CD45 protein tyrosine phosphatase is a potent immunomodulatory target, the mechanisms by which antibody against CD45RB isoforms (anti-CD45RB) induces allograft tolerance remain unclear. We show here that anti-CD45RB treatment alters CD45 isoform expression on T cells, which is associated with rapid up-regulation of CTLA-4 expression. These effects appear specific and occur without up-regulation of other activation markers. Administration of a blocking monoclonal antibody to CTLA-4 at the time of transplantation prevents anti-CD45RB therapy from prolonging islet allograft survival. In addition, treatment with cyclosporin A blocks anti-CD45RB-induced CTLA-4 expression and promotes acute rejection. These data suggest that anti-CD45RB acts through mechanisms that include CTLA-4 up-regulation and demonstrate a link between CD45 and CTLA-4 that depends on calcineurin-mediated signaling. They demonstrate also that CTLA-4 expression may be specifically targeted to enhance allograft acceptance.

Abatacept↗

PubMed2001

P-113D, an antimicrobial peptide active against Pseudomonas aeruginosa, retains activity in the presence of sputum from cystic fibrosis patients.

Antimicrobial peptides are a source of novel agents that could be useful for treatment of the chronic lung infections that afflict cystic fibrosis (CF) patients. Efficacy depends on antimicrobial activity against the major pathogens of CF patients, Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in the environment of the CF patient's airway. We describe the in vitro efficacies of derivatives of histatins, which are histidine-rich peptides produced by the salivary glands of humans and higher primates. P-113, a peptide containing 12 of the 24 amino acid residues of the parent molecule, histatin 5, retained full antibacterial activity and had a good spectrum of activity in vitro against the prominent pathogens of CF patients. However, P-113 was not active in the presence of purulent sputum from CF patients. In contrast, P-113D, the mirror-image peptide with the amino acid residues in the D configuration, was stable in sputum, was as active as P-113 against pathogens of CF patients in the absence of sputum and retained significant activity in the presence of sputum from CF patients. Recombinant human DNase, which effectively liquefies sputum, enhanced the activity of P-113D in undiluted sputum against both exogenous (added) bacteria and endogenous bacteria. Because of its properties, P-113D shows potential as an inhalant in chronic suppressive therapy for CF patients.

Anti-Bacterial Agents↗

PubMed2001

Anticandida activity is retained in P-113, a 12-amino-acid fragment of histatin 5.

Through the analysis of a series of 25 peptides composed of various portions of the histatin 5 sequence, we have identified P-113, a 12-amino-acid fragment of histatin 5, as the smallest fragment that retains anticandidal activity comparable to that of the parent compound. Amidation of the P-113 C terminus increased the anticandidal activity of P-113 approximately twofold. The three histidine residues could be exchanged for three hydrophobic residues, with the fragment retaining anticandidal activity. However, the change of two or more of the five basic (lysine and arginine) residues to uncharged residues resulted in a substantial loss of anticandidal activity. A synthetic D-amino-acid analogue, P-113D, was as active against Candida albicans as the L-amino-acid form. In vitro MIC tests in low-ionic-strength medium showed that P-113 has potent activity against Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis. These results identify P-113 as a potential antimicrobial agent in the treatment of oral candidiasis.

Amino Acid Sequence↗

PubMed2000

Sustained release of silver from periodontal wafers for treatment of periodontitis.

Periodontal wafers intended to treat the underlying infections in patients with periodontitis have been developed. The wafers consist of poly(lactic-co-glycolic acid) as a primary bioerodible polymeric component, poly(ethylene glycol) as a plasticizer and encapsulation aid, and silver nitrate as the antimicrobial agent. The wafers are capable of sustained in vitro release of bioactive silver for at least 4 weeks. The wafers exhibit silver release that follows erosion kinetics, confirming a bulk erosion/release mechanism. In clinical evaluation, sustained release of silver at bactericidal levels for at least 21 days is observed. Staining of hard and soft tissues due to the released silver is minimal and reversible.

Administration, Buccal↗

PubMed2000

Photoactive porphyrin derivative with broad-spectrum activity against oral pathogens In vitro.

Photodynamic therapy (PDT) has historically been used as a means to treat cancerous tumors but has recently been used to kill bacterial cells through the use of targeted photosensitizers. PDT is a potential adjunct to scaling and root planing in the treatment of periodontal disease. However, the effectiveness of porphyrin derivatives against microorganisms has been limited because some gram-negative bacteria are refractory to photodynamic treatment with these agents. We have designed a porphyrin derivative conjugated to a pentalysine moeity that endows the molecule with activity against gram-positive and gram-negative bacteria. Whereas the porphyrin, chlorin e6, showed in vitro activity against a limited spectrum of bacteria, chlorin e6 conjugated to pentalysine showed in vitro activity against all oral microorganisms tested, including Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. polymorphum, Actinomyces viscosus, and the streptococci. Potent antimicrobial activity (>/=5-log-unit reduction in the numbers of CFU per milliliter) was retained in the presence of up to 25% whole sheep blood. The use of potent, selective agents such as this chlorin e6-pentalysine conjugate to more effectively reduce the pathogenic bacteria in the periodontal pocket may be a significant tool for the treatment of periodontal disease.

Aggregatibacter actinomycetemcomitans↗

PubMed1998

The second domain of the CD45 protein tyrosine phosphatase is critical for interleukin-2 secretion and substrate recruitment of TCR-zeta in vivo.

The CD45 protein tyrosine phosphatase (PTPase) has been shown to regulate the activity of Lck and Fyn protein tyrosine kinases in T cells. However, it is not clear that these constitute the only CD45 substrates. Moreover, the manner by which PTPase activity and substrate recruitment are regulated, is poorly understood. Previous in vitro studies suggest that the first cytoplasmic PTPase domain (D1) of CD45 is the active PTPase, which may be regulated by an enzymatically inactive second PTPase domain (D2). However, the function of CD45 D2 in vivo is unknown. In this study, reconstitution of CD45(-) T cells with specific CD45 PTPase mutants allowed demonstration of a critical role for D2 in TCR-mediated interleukin (IL)-2 production. Specifically, replacement of CD45 D2 with that of the LAR PTPase to form a CD45/LAR:D2 chimera, abrogates CD45-dependent IL-2 production. This effect cannot be accounted for by loss of PTPase activity per se. The expression of D1 substrate-trapping mutants reveals an in vivo interaction between CD45 and TCR-zeta that is dependent on CD45 D2. Thus, cells expressing CD45 lacking D2 exhibit abnormal TCR-mediated signaling characterized by hyperphosphorylation of zeta and deficient ZAP-70 phosphorylation. These data suggest an essential role for CD45 D2 in TCR-regulated IL-2 production through substrate recruitment of the zeta chain.

Humans↗

PubMed1998

Antibody-mediated targeting of CD45 isoforms: a novel immunotherapeutic strategy.

CD45 is a family of transmembrane protein tyrosine phosphatases exclusively expressed by hematopoietic cells and critically involved in the regulation of T cell activation signals. We now demonstrate that three 100-microg doses of anti-CD45RB mAb MB23G2 can induce long-term engraftment of islets into major histcompatibility complex-disparate chemically diabetic mice. Long-term graft survivors (>120 days) were tolerant to new islet allografts from the original donor strain. MB23G2 induced a temporary decrease in number circulating leukocytes but had no effect on leukocyte number in other lymphoid compartments. Histologic examination of allografts from treated and untreated recipients revealed a similar peri-islet infiltration on day 6. Eleven days after transplant, the peri-islet infiltrate in treated animals persisted, but in marked contrast to untreated control animals, there was no insulitis and islet integrity was preserved. The peri-islet infiltrate from treated animals showed a mild increase in CD4 cells, a decrease in CD8 cells, and decreased intensity of CD45RB expression. Treatment of naive animals with anti-CD45RB (MB23G2) resulted in a shift in CD45 isoform expression on T cells with a loss of higher molecular weight isoforms and increased expression of lower molecular weight (CD45R0) isoform. This shift in CD45 isoform expression from CD45RBHi to CD45RBLo was associated with an increase in the intragraft expression of transcripts for interleukin (IL) 4 and IL-10, consistent with the expected activity of this distinct immunoregulatory T cell subset. Antibody-mediated targeting of CD45 may induce tolerance through novel mechanisms and have direct applicability to clinical transplantation in humans.

Animals↗

PubMed1998

Screening systems for detecting inhibitors of cell wall transglycosylation in Enterococcus. Cell wall transglycosylation inhibitors in Enterococcus.

We devised two screening systems to detect cell wall transglycosylation inhibitors. One screen utilizes a mutant of Enterococcus faecalis strain A256 that is dependent on vancomycin or moenomycin for growth. In the absence of transglycosylation inhibitors the strain fails to grow, while in the presence of inhibitors, cells are rescued. A second screening organism E. faecalis strain MDD212 utilizes a translational fusion of the lacZ gene to the vanH promoter in a derivative of E. faecalis that contains a vancomycin resistance determinant. Induction of beta-galactosidase occurs when cells are exposed to inhibitors of transglycosylation. Our natural products drug source of fungal fermentations was tested with these screens. Several cultures that produced the same family of compounds, called the thielavins, were detected. Thielavin B inhibited the formation of peptidoglycan in an in vitro assay, suggesting that these screening systems can detect compounds that interfere with cell wall transglycosylation.

Base Sequence↗

PubMed1997

Indefinite islet allograft survival in mice after a short course of treatment with anti-CD45 monoclonal antibodies.

BACKGROUND: Although islet cell transplantation is considered an ideal form of endocrine replacement for type I diabetes, clinical application in humans is still not feasible. New immunosuppressive strategies are clearly needed to control inexorable rejection. CD45 is a family of transmembrane protein tyrosine phosphatases critically involved in the regulation of lymphocyte activation signals. Anti-CD45RB monoclonal antibody can prevent rejection of murine renal allografts. METHODS: Here, we examine the consequences of targeting CD45 in murine islet cell transplantation. Diabetic mice recipients received islet allografts under the kidney capsule and were divided into seven groups. Recipients received no treatment (controls) or anti-CD45RB monoclonal antibody (mAb; MB23G2 or C363.16A) at different dosages and treatment intervals. RESULTS: All untreated control animals lost islet function, becoming hyperglycemic within 10-17 days after transplantation. Animals treated with either anti-CD45RB mAb showed a significant prolongation of islet allograft survival when compared with controls. Anti-CD45RB MB23G2 at 100 microg/day, given on days -1, 0, and 5 was particularly effective, inducing indefinite islet allograft survival in 60% of recipients. CONCLUSIONS: These results indicate that anti-CD45 mAbs are potent immunomodulatory agents, able to sustain indefinite islet allograft function after a short treatment course in the highly immunogenic model of islet transplantation.

Animals↗

PubMed1996

Differential regulation of activation-induced tyrosine phosphorylation and recruitment of SLP-76 to Vav by distinct isoforms of the CD45 protein-tyrosine phosphatase.

The CD45 family of transmembrane protein-tyrosine phosphatases plays a critical role in T cell activation signaling by regulating the tyrosine phosphorylation of protein-tyrosine kinases and their substrates. Multiple alternatively spliced CD45 isoforms, differing only in their extracellular domains, are differentially expressed by subsets of T cells with distinct functional repertoires. However, the physiological function of the various isoforms remains elusive. Using a novel panel of Jurkat T cell clones that uniquely express either the smallest (CD45(0)) or the largest (CD45(ABC)) isoform, we previously demonstrated CD45 isoform-specific differences in interleukin-2 secretion and tyrosine phosphorylation of Vav. We now demonstrate differential activation-induced tyrosine phosphorylation of a 76-kDa Vav-associated protein (pp76) by cells expressing distinct CD45 isoforms. The tyrosine phosphorylation of Vav and associated pp76 follow parallel kinetics. pp76 interacts with the SH2 and SH3 domains of Vav. We have identified pp76 as SLP-76, a recently cloned Grb2-binding protein. After activation with anti-CD3, CD45(ABC) transfectants demonstrate increased tyrosine phosphorylation and physical association of SLP-76 with Vav compared to transfectants expressing CD45(0). These results establish a novel physical link between Vav and SLP-76 that is differentially regulated by CD45 isoform expression.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Distinct isoforms of the CD45 protein-tyrosine phosphatase differentially regulate interleukin 2 secretion and activation signal pathways involving Vav in T cells.

The CD45 family of transmembrane protein-tyrosine phosphatases plays a crucial role in the regulation of lymphocyte activation by coupling activation signals from antigen receptors to the signal transduction apparatus. Multiple CD45 isoforms, generated through regulated alternative mRNA splicing, differ only in the length and glycosylation of their extracellular domains. Differential distribution of these isoforms defines subsets of T cells having distinct functions and activation requirements. While the requirement for the intracellular protein-tyrosine phosphatase domains has been documented, the physiological role of the extracellular domains remains elusive. Here we report the generation of CD45-antisense transfected Jurkat T cell clones that lack CD45 or have been reconstituted to uniquely express either the smallest, CD45(0), or the largest, CD45(ABC), isoform. These cells exhibited marked isoform-dependent differences in IL-2 production and tyrosine phosphorylation of cellular proteins, including Vav after anti-CD3 stimulation. These results demonstrate that the distinct CD45 extracellular domains differentially regulate T cell receptor-mediated signaling pathways. Furthermore, these findings suggest that alterations in CD45 isoform expression by individual T cells during thymic ontogeny and after antigen exposure in the periphery directly affects the signaling pathways utilized.

Animals↗

PubMed1995

Genetic analysis suggests functional interactions between the N- and C-terminal domains of the TetA(C) efflux pump encoded by pBR322.

Genetic analysis of the tetA(C) gene of pBR322 indicates the importance of two-cytoplasmic loops in the TetA(C) protein (P. McNicholas, I. Chopra, and D. M. Rothstein, J. Bacteriol. 174:7926-7933, 1992). In this study, we characterized second-site suppressor mutations that suggest a functional interaction between these two cytoplasmic regions of the protein.

Amino Acid Sequence↗

PubMed1994

Mutations in the tetA(B) gene that cause a change in substrate specificity of the tetracycline efflux pump.

The tetA(B) gene from transposon Tn10 fails to mediate resistance to the novel tetracycline analog 9-(dimethylglycylamido)minocycline (DMG-Mino) (P. E. Sum, V. J. Lee, R. T. Testa, J. J. Hlavka, G. A. Ellestad, J. D. Bloom, Y. Gluzman, and F. P. Tally, J. Med. Chem. 37:184-188, 1994; R. T. Testa, P. Petersen, N. V. Jacobus, P. E. Sum, V. J. Lee, and F. P. Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). Mutations in either of two codons of tetA(B) that resulted in increased resistance to DMG-Mino also caused diminished resistance to tetracycline, identifying amino acid residues critical for the recognition of tetracycline.

Amino Acid Sequence↗

PubMed1993

The tet(K) gene of plasmid pT181 of Staphylococcus aureus encodes an efflux protein that contains 14 transmembrane helices.

The corrected DNA sequence of the tet(K) structural gene, encoding the tetracycline efflux protein from Staphylococcus aureus, is reported. The Tet(K) protein is homologous to related tetracycline efflux proteins throughout the protein, including at the C-terminal end. Hydropathy plotting now clearly indicates that the Tet(K) protein contains 14 transmembrane alpha-helices.

Amino Acid Sequence↗

PubMed1993

Human CD4/CD45RA+ and CD4/CD45RA- T cell subsets express CD4-p56lck complexes, CD4-associated lipid kinases, TCR/CD3-p59fyn complexes, and share similar tyrosine kinase substrates.

T cell activation appears to be regulated by an interplay between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). p56lck and p59fyn have been found to associate with CD4 and TCR-CD3 respectively. The CD45 family of transmembrane PTPases has been shown to be able to regulate the activities of these receptor-associated PTKs in vitro. In man, CD45 contains five different isoforms whose distribution defines subsets of T cells having distinct activation requirements and in vitro functions. Several groups have reported a physical interaction between distinct isoforms of CD45 and CD2, CD4, and the TCR-CD3 complex. Given the potential regulatory interaction between CD45 and PTKs in CD4+ subsets expressing different CD45 isoforms, we have examined CD4 associated and TCR-CD3- associated PTK activities, associated phosphatidyl inositol (PI) kinases and substrates of tyrosine phosphorylation in CD45RA+ and CD45RA- CD4+ T cell lines derived from peripheral blood. Both subsets express CD4-associated p56lck and TCR-CD3-associated p59fyn kinases which exhibit identical in vitro phosphorylation at the Y-394 and Y-420 autophosphorylation sites respectively. Further, both subsets exhibited PI kinases activity associated with CD4-p56lck. Consistent with these observations, anti-CD3 crosslinking induced the phosphorylation of a similar spectrum of intracellular substrates in these CD45RA+ and CD45RA- CD4+ T cell lines. These observations indicate that despite the possible interaction between CD45 isoforms and CD4 or TCR-CD3, the mere expression of the CD45RA isoform does not in and of itself alter the presence of receptor-associated kinases or their intracellular targets.

1-Phosphatidylinositol 4-Kinase↗

PubMed1993

Expression of the tetK gene from Staphylococcus aureus in Escherichia coli: comparison of substrate specificities of TetA(B), TetA(C), and TetK efflux proteins.

The tetK gene, which encodes a tetracycline efflux pump from Staphylococcus aureus, was expressed in Escherichia coli by using an inducible, low-level expression system. The tetK gene, as well as the tetA(B) gene from the transposon Tn10 and the tetA(C) gene from plasmid pBR322, was subjected to the regulatory control of the lac repressor, and resistance to tetracycline was measured as a function of the isopropyl-beta-D-thiogalactopyranoside concentration. The maximum resistance of the E. coli strain containing the tetK construct was comparable to the maximum resistance of the strain containing the tetA(C) construct but was less than the resistance of the strain containing the tetA(B) construct. Overexpression of the tetK, tetA(B), or tetA(C) genes was toxic. When expression was regulated so that resistance to tetracycline was comparable, then the TetA(B) and TetA(C) proteins conferred very similar levels of resistance to a variety of tetracycline derivatives. In contrast, the TetK protein was less capable of conferring resistance to the tetracycline derivatives minocycline, 6-deoxy-6-demethyltetracycline, and doxycycline. The implications for the recognition of various tetracycline substituents by the TetK protein are discussed.

Bacterial Proteins↗