Tildrakizumab (MK-3222), an anti-interleukin-23p19 monoclonal antibody, improves psoriasis in a phase IIb randomized placebo-controlled trial.

BACKGROUND: Tildrakizumab is a high-affinity, humanized, IgG1/κ, anti-interleukin (IL)-23p19 monoclonal antibody that does not bind human IL-12 or p40 is being developed for the treatment of chronic plaque psoriasis. OBJECTIVES: To evaluate the safety and efficacy of subcutaneous tildrakizumab in patients with moderate-to-severe chronic plaque psoriasis. METHODS: A three-part, randomized, double-blind, phase IIb trial was conducted in 355 adults with chronic plaque psoriasis. Participants were randomized to receive subcutaneous tildrakizumab (5, 25, 100, 200 mg) or placebo at weeks 0 and 4 (part I) and every 12 weeks thereafter until week 52 (part II). Study drug was discontinued at week 52 and participants were followed through week 72 (part III). Primary efficacy end point was Psoriasis Area and Severity Index (PASI) 75 response at week 16. Adverse events (AEs) and vital signs were monitored throughout the study. RESULTS: At week 16, PASI 75 responses were 33·3% (n = 14), 64·4% (n = 58), 66·3% (n = 59), 74·4% (n = 64) and 4·4% (n = 2) in the 5-, 25-, 100- and 200-mg tildrakizumab and placebo groups, respectively (P ≤ 0·001 for each tildrakizumab dose vs. placebo). PASI 75 response was generally maintained through week 52; only eight of 222 participants who achieved PASI 75 response at week 52 and continued to part III relapsed following discontinuation up to week 72. Possible drug-related serious AEs included bacterial arthritis and lymphoedema (part I), and melanoma, stroke, epiglottitis and knee infection (part II). CONCLUSIONS: Tildrakizumab had treatment effects that were superior to placebo, maintained for 52 weeks of treatment, and persisted for 20 weeks after cessation. Tildrakizumab was generally safe and well tolerated. These results suggest that IL-23p19 is a key target for suppressing psoriasis.

Interleukin-23 is sufficient to induce rapid de novo gut tumorigenesis, independent of carcinogens, through activation of innate lymphoid cells.

Chronic inflammation has been associated with increased risk for developing gastrointestinal cancer. Interleukin-23 (IL-23) receptor signaling has been correlated with inflammatory bowel disease pathogenesis, as well as promotion of tumor growth. However, little is known about the relative potential for IL-23-directed causality in gut tumorigenesis. We report that IL-23 transgene expression was sufficient to induce rapid (3-4 weeks) de novo development of intestinal adenomas with 100% incidence. Initiation of tumorigenesis was independent of exogenous carcinogens, Helicobacter colonization, or pre-existing tumor-suppressor gene mutations. Tumorigenesis was mediated by Thy1(+)IL-23R(+) innate lymphoid cells (ILC3), in part, through IL-17 responses as tumor development was inhibited in RAG(-/-) × IL-17(-/-) double knockout mice. Remarkably, IL-23 initiation of tumorigenesis by resident ILCs consistently occurred before recruitment of conspicuous inflammatory infiltrates. Our results reveal an explicit role for IL-23-mediated initiation of gut tumorigenesis and implicate a key role for IL-23R(+) ILC3 in the absence of overt cellular infiltrate recruitment.

Developmental switches in chemokine response profiles during B cell differentiation and maturation.

Developing B cells undergo dramatic changes in their responses to chemoattractant cytokines (chemokines) and in expression of chemokine receptors. Bone marrow pre-pro-B cells (AA4.1(+)/natural killer 1.1(-) Fraction A cells) and cells capable of generating pro-B colonies in the presence of interleukin 7 and flt3 ligand migrate to thymus-expressed chemokine (TECK), a response lost in later stages of B cell development. B cell-attracting chemokine 1 (BCA-1) responses correlate with CXC chemokine receptor (CXCR)5 expression, are first displayed by a pro-B cell subset, are lost in pre-B cells, and then are regained just before and after egress from the marrow. All peripheral B cell subsets, including follicular and germinal center as well as marginal zone and peritoneal B1 B cells, respond to BCA-1, implying that responsiveness to this follicular chemokine is not sufficient to predict follicle localization. Responses to the CC chemokine receptor (CCR)7 ligands secondary lymphoid tissue chemoattractant (SLC) and macrophage inflammatory protein (MIP)-3beta, implicated in homing to lymphoid tissues, are upregulated before B cell exit from the marrow, but increase further in the periphery and are shared by all peripheral B cells. In contrast, responsiveness to MIP-3alpha and expression of CCR6 are acquired only after emigration to the periphery and during maturation into the recirculating B cell pool. Chemotaxis to stromal cell-derived factor 1alpha is observed at all stages of B cell differentiation. Thus, unique patterns of chemokine responses may help define developing B cell populations and direct their maturation in the marrow and migration to the periphery.

Regulation of chemotactic and proadhesive responses to chemoattractant receptors by RGS (regulator of G-protein signaling) family members.

Serpentine Galphai-linked receptors support rapid adhesion and directed migration of leukocytes and other cell types. The intracellular mechanisms mediating and regulating chemoattractant-directed adhesion and locomotion are only now beginning to be explored. RGS (for regulator of G-protein signaling) proteins are a recently described family that regulate Galphai-stimulated pathways by acting as GTPase-activating proteins. Little is known about the GTPase activity of the Galphai proteins involved in adhesion and chemotaxis, or the significance of their regulation to these responses. Using transiently transfected lymphoid cells as a model system, we show that expression of RGS1, RGS3, and RGS4 inhibits chemoattractant-induced migration. In contrast, RGS2, a regulator of Galphaq activity, had no effect on cell migration to any chemoattractant. RGS1, RGS3, and RGS4 also reduced rapid chemoattractant-triggered adhesion, although the proadhesive response appears quantitatively less sensitive to RGS action than chemotaxis. The results suggest that the duration of the Galphai signal may be a particularly important parameter in the chemotactic responses of leukocytes, and demonstrate the potential for RGS family members to regulate cellular adhesive and migratory behaviors.

6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3beta receptor CCR7.

The beta chemokine known as 6-C-kine, secondary lymphoid-tissue chemokine (SLC), TCA4, or Exodus-2 (herein referred to as 6CK/SLC) can trigger rapid integrin-dependent arrest of lymphocytes rolling under physiological shear and is highly expressed by high endothelial venules, specialized vessels involved in lymphocyte homing from the blood into lymph nodes and Peyer's patches. We show that 6CK/SLC is an agonist for the lymphocyte chemoattractant receptor, CCR7 (EBI-1, BLR-2), previously described as a receptor for the related beta chemokine MIP-3beta (ELC or Exodus-3). Moreover, 6CK/SLC and MIP-3beta attract the same major populations of circulating lymphocytes, including naive and memory T cells > B cells (but not natural killer cells); desensitization to MIP-3beta inhibits lymphocyte chemotaxis to 6CK/SLC but not to the alpha chemokine SDF-1 (stromal cell-derived factor); and 6CK/SLC competes for MIP-3beta binding to resting mouse lymphocytes. The findings suggest that the majority of circulating lymphocytes respond to 6CK/SLC and MIP-3beta in large part through their common receptor CCR7 and that these molecules may be important mediators of physiological lymphocyte recirculation in vivo.

ADP-ribosylation factor functions synergistically with a 50-kDa cytosolic factor in cell-free activation of human neutrophil phospholipase D.

Proteins in both the cytosol and plasma membrane are needed to reconstitute cell-free phospholipase D activity from phagocytes (Olson, S., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242); membrane factors include a small GTP-binding protein in the Rho family (Bowman, E., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512). ADP-ribosylation factor (ARF) was recently implicated as the cytosolic factor, as it activates phospholipase D in HL-60 membranes. Herein, we show that ion exchange chromatography separates ARF from the major phospholipase D-stimulating cytosolic factor. Both bovine brain ARF and recombinant human ARF-1 stimulated a small amount of phospholipase D activity in the absence of cytosol (about 10% of the response seen with cytosol). With a high concentration of ARF-depleted cytosol, ARF did not further activate. However, at low cytosol, ARF caused marked activation. Thus, ARF synergizes with the cytosolic factor in phospholipase D activation.

Neutrophil phospholipase D is activated by a membrane-associated Rho family small molecular weight GTP-binding protein.

Phospholipase D in human neutrophil lysates is activated by GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)), implying the participation of a GTP-binding protein. Reconstitution of GTP gamma S-dependent activity requires protein factors in both the plasma membrane and the cytosol (Olson, S. C., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242). The location of the GTP-binding protein was investigated by preincubating either cytosol or plasma membrane with GTP gamma S, followed by removal of all but tightly bound nucleotide and reconstituting activity with the complementing untreated subcellular fraction. This approach indicated that the GTP-binding protein was membrane-associated. A low magnesium requirement for GTP gamma S prebinding, as well as a failure of aluminum fluoride to activate, suggested a Ras-like small M(r) GTP-binding protein. smg GDP dissociation stimulator, which stimulates the exchange of GDP for GTP on a variety of small GTP-binding proteins, stimulated GTP-dependent phospholipase D activity. Rho GDP dissociation inhibitor, a regulatory protein that binds specifically to and inhibits the functions of Rho family small GTP-binding proteins, inhibited GTP gamma S-dependent activity. Thus, neutrophil phospholipase D is regulated by a membrane-associated small molecular weight GTP-binding protein, likely to be a member of the Rho family.

Phospholipase D activation in a cell-free system from human neutrophils by phorbol 12-myristate 13-acetate and guanosine 5'-O-(3-thiotriphosphate). Activation is calcium dependent and requires protein factors in both the plasma membrane and cytosol.

Receptor-linked activation of phospholipase D has been demonstrated recently in a variety of intact cell types including granulocytes, but little is known about the enzyme, its cofactor requirements, and regulation. Using [3H]alkyllysophosphatidylcholine to prelable an endogenous phosphatidylcholine substrate pool in conjunction with transphosphatidylation using ethanol to generate labeled phosphatidylethanol, we demonstrated a novel phospholipase D activity in neutrophil subcellular fractions. Guanosine 5'-O-3-(thiotriphosphate) (GTP gamma S) and phorbol 12-myristate 13-acetate (PMA) activated both phosphatidic acid generation and transphosphatidylation. Activity using both activators required the presence of not only plasma membrane but also cytosol, and proteolytic and thermal inactivation demonstrated the requirement for protein factors in both fractions. Using both stimuli, activity increased with increasing cytosol concentration. Product formation was approximately linear for about 10 min with PMA and 30 min with GTP gamma S, and both activators resulted in the total hydrolysis of up to 10% of the labeled phosphatidylcholine. The activity using both activators showed similar broad neutral pH optima, and both required the presence of micromolar levels of calcium, which by itself failed to activate at concentrations up to 1 mM. At low micromolar concentrations of nucleotides, activation was specific for guanine nucleotides and showed a specificity of GTP gamma S greater than guanyl-5'-yl imidodiphosphate greater than GTP, with no effect of GDP and GMP or adenine nucleotides, consistent with the participation of a guanine nucleotide regulatory protein. PMA activation was dependent on the presence of ATP, in particular when dialyzed cytosol was used, and was inhibited by about 50% by staurosporine, supporting a role for protein kinase C. However, purified protein kinase C failed to substitute for cytosol, implicating an additional cytosolic factor(s) in this response. These results indicate that the granulocytic phospholipase D pathway is a complex system that is regulated by at least two activation pathways, each comprised of components in two subcellular compartments.

Stimulation of adrenal mitochondrial cholesterol side-chain cleavage by GTP, steroidogenesis activator polypeptide (SAP), and sterol carrier protein2. GTP and SAP act synergistically.

Several factors are known to stimulate cholesterol side-chain cleavage in isolated adrenal mitochondria, including steroidogenesis activator polypeptide (SAP), GTP, and sterol carrier protein2 (SCP2). All of these reportedly function at the level of the translocation of cholesterol to the inner membrane wherein side-chain cleavage to form pregnenolone occurs. We have investigated the activating effects of these factors alone and in combination. Under conditions where exogenous cholesterol is provided and multiple turnovers of a transport system are required, GTP stimulated steroidogenesis in isolated mitochondria and in adrenal homogenates, and this effect was enhanced by a GTP regenerating system. SAP alone had little effect under these conditions, but synergized with GTP to stimulate cholesterol metabolism. A truncated SAP analog and a variant from the C terminus of the minor heat-shock protein GRP78 had similar effects, but an unrelated peptide had no effect. GTP stimulated side-chain cleavage with the same EC50 in both resting mitochondria (from dexamethasone-treated rats) and in activated mitochondria (from ether-treated rats), but SAP effects were most apparent in resting mitochondria. In contrast, SCP2 stimulation was additive with other factors, suggesting an independent mechanism of action. While the data are consistent with biological roles for these factors, the relatively small magnitude of the in vitro effects may indicate that cell disruption and mitochondrial isolation disrupt important structural or other features which are necessary for the full expression of the steroidogenic response.

Chemical synthesis, initial conformational studies, and activity of rat steroidogenesis activator peptide and a truncated analog.

A 30-residue peptide corresponding to the amino acid sequence of steroidogenesis activator peptide (SAP) from rat Leydig tumor cells has been synthesized by the solid-phase method using Boc protection. SAP is the putative cycloheximide-sensitive, cAMP-regulated mediator of ACTH-stimulated conversion of cholesterol to pregnenolone in adrenal cortex. N alpha-acetyl-SAP(11-30), an NH2-terminally truncated steroidogenesis activator peptide analog that is missing the most hydrophobic portion of SAP, was also prepared. In addition to these two peptides, N alpha-acetyl-(Cys0)SAP was synthesized in both non-radiolabeled and tritiated forms for coupling to carrier proteins for use as an immunogen to raise anti-SAP antibodies. Chain elongation during synthesis of SAP on PAM resin proceeded with an average coupling yield of 99.3% as determined by quantitative ninhydrin tests. After HF cleavage at -7 degrees, the crude products were purified by semi-preparative HPLC. Peptides were analyzed by analytical HPLC, amino acid analysis, tryptic peptide mapping, and by UV and CD spectroscopy. As determined by CD spectra, SAP showed little evidence of preferred structure either in aqueous solution in the presence of divalent cations or in micelles of reduced Triton X-100 in the absence or presence of either cholesterol or phosphatidylcholine. SAP, in conjunction with GTP, enhanced side chain cleavage activity in isolated adrenal mitochondria.

Asperger's syndrome and autism: the case for a connection.

There is considerable disagreement on the classification of Asperger's syndrome and its relationship to autism. Unlike autism, Asperger's is not usually recognised before 30 months of age, and speech delay is not typical. However, if a child exhibits all the symptoms of autism apart from these two features, are these sufficient grounds for withholding a diagnosis of autism? This paper describes four boys and their father who, by presenting at various points on the autistic/Asperger spectrum, embody this dilemma for the diagnostician. The implications for research and clinical practice are discussed.

Selective toxicity of deoxyadenosine analogues in human melanoma cell lines.

The in vitro toxicities of 19 analogues of deoxyadenosine were tested using a panel of human melanoma cell lines including two lines sensitive to deoxyadenosine and deoxyinosine. The 2-fluoro-, 2-chloro-, 2-bromo- and 2-amino-8-aza derivatives were the most toxic and showed selectivity against deoxyadenosine-sensitive cells. 2-Bromodeoxyadenosine (BrdAdo) and its 5'-phosphate were less potent than the chloro compound but showed the greatest selectivity. In further studies of BrdAdo a third sensitive melanoma line was identified of the eight tested. A treatment time of 24 hr or more was required to develop toxicity to BrAdo; this could be prevented by deoxycytidine or cytidine added to the medium but not by other nucleosides. Flow cytometry showed that BrdAdo blocked cells in the G1 and S phases of the cell cycle. DNA synthesis as judged by thymidine incorporation was rapidly inhibited by BrdAdo to an extent which reflected the sensitivity of the particular cell line; RNA synthesis was less affected. Exposure to BrdAdo for 48 hr induced breaks in the preformed DNA of sensitive but not resistant cells. The results suggest that the toxicity of BrdAdo is associated with prolonged inhibition of DNA synthesis and subsequent DNA fragmentation.

A comparison of cobalamin binding by liver and kidney in rat and man.

Binding of cobalamin (Cbl) was compared in liver and kidney plasma membranes prepared from rat and human tissues. Single, high-affinity, saturable (200 pmol/l), binding sites for TC II-Cbl were found in all tissues; by contrast no receptors were present for free cobalamin, for which only non-specific adsorption occurred. Binding constants for TC II-CNCbl determined for liver and kidney plasma membranes were of a similar magnitude. Mean values for Ka (litre/nmol) were 16.7 (rat liver), 18.8 (rat kidney), 8.0 (human liver) and 7.5 (human kidney). Results for binding TC II-OHCbl instead of TC II-CNCbl showed no difference in Ka and Bmax. values, although the non-specific adsorption was decreased to a third. Competitive inhibition results showed that the receptors are specific for the TC II molecule and that binding is unaffected either by the cobalamin moiety or by the presence of free cobalamin. Degradation of the receptor protein molecules by trypsin (10 micrograms/ml) resulted in 90% inhibition of binding. It is concluded that differences between liver and kidney in cobalamin uptake and accumulation cannot be attributed to differences in their TC II receptors.

The regulatory roles of liver and kidney in cobalamin (vitamin B12) metabolism in the rat: the uptake and intracellular binding of cobalamin and the activity of the cobalamin-dependent enzymes in response to varying cobalamin supply.

To examine possible regulatory roles of liver and kidney in cobalamin metabolism, specific activities of the two cobalamin-dependent enzymes, uptake in vivo of cyano [57Co]cobalamin [( 57Co]CNCbl) and the binding of [57Co]Cbl to intracellular proteins were measured in normal, cobalamin-loaded and cobalamin-deficient rats. Cobalamin deficiency and cobalamin loading produced greater changes in cobalamin concentration in the kidney than in the liver. Although cobalamin deficiency resulted in a decrease in total methylmalonyl-coenzyme A mutase (methylmalonyl-CoA mutase) in both organs, cobalamin loading had no effect. Neither deficiency nor loading altered total methyltransferase activity. The holoenzyme activities of both enzymes correlated with changes in tissue cobalamin levels. Uptake of [57Co]Cbl indicated that the kidney, in contrast to the liver, increased its uptake during loading and reduced it during deficiency, suggesting a possible regulatory role for this organ. In the normal rat, 24 h after injection of [57Co]CNCbl, 0.3% of the administered [57Co]Cbl was present in the liver as free cobalamin. By contrast, in the kidney, over 13% of the [57Co]Cbl was present in the free form. During deficiency free renal [57Co]Cbl was reduced to 0.6% of the administered [57Co]Cbl whereas in cobalamin-loaded rats it was increased to more than 27%. It is concluded that alterations in tissue cobalamin levels resulting from differences in cobalamin supply are due to changes in the large pool of free cobalamin present in the kidney and not to changes in the intracellular binding.

Biofeedback as an adjunct to psychotherapy in the treatment of vaginismus.

This exploratory study examines the use of biofeedback as an adjunct to psychotherapy in the treatment of vaginismus. A set of six Sims-type graded EMG probes was constructed to provide biofeedback from the vaginal sphincter and was tested on a pilot sample of nulliparous women prior to this study. Five sequential cases of vaginismus from a clinic waiting list participated in the program. All five couples completed the program and all reported successful intercourse at its conclusion. The number of sessions devoted to biofeedback probe insertion was almost halved in comparison to previous experience with Sims dilators. Overall treatment duration was not shortened. At follow-up 6 months later, two couples reported pregnancy, one couple was having regular intercourse, and two couples had ceased intercourse. The authors conclude that biofeedback is an effective aid to learning muscle control, is acceptable to patients, and may increase the success rate by minimizing dropouts. The importance of follow-up is stressed.

Day patient versus in-patient: factors determining selection of acutely ill patients for hospital treatment.

In a prospective study the criteria and characteristics associated with the admission of acutely ill psychiatric patients to in-patient or day hospital care were examined. Over a four-month period, 54 patients were admitted to hospital and 43 to a day hospital. There was significantly more schizophrenics in the hospital. Day hospital patients were significantly younger, had shorter psychiatric histories, were considered less severely ill and had more insight into their illness. Hospital patients had poorer employment histories, and perceived their families as less supportive; admission had more often been requested by them or their families.