[T-peptide Enhances the Killing Effects of Cisplatinum on Lung Cancer].

BACKGROUND: T peptide is extensively used in anti-tumor treatment. The aims of this study were to investigate whether T peptide enhances cisplatinum efficiency while reducing its side effects and to identify its effective mechanisms. METHODS: (1) Human macrophage U937 cells were treated with T peptide and/or cisplatinum. The levels of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) of each group were detected by enzyme-linked immunosorbent assay (ELISA); (2) Xenograft mouse models of human lung cancer were treated with T peptide and/or cisplatinum once every five days for three times. Tumor volumes were measured during treatment; (3) The percentages of macrophages in the peripheral blood of the xenograft mouse models were measured by FACS. RESULTS: (1) Compared with other groups, the level of TNF-α was significantly higher in the human macrophage U937 cells that were treated with T peptide combined with cisplatinum. The levels of IFN-γ were significantly higher in human macrophage U937 cells that were treated with T peptide alone or T peptide combined with cisplatinum; (2) In the xenograft mouse models, T peptide combined with cisplatinum treatment significantly inhibited tumor growth without weight loss compared with the other groups; (3) The percentages of macrophages in the peripheral blood were significantly higher in the xenograft mouse models that were treated with T peptide combined with cisplatinum compared with in the other groups. CONCLUSIONS: T peptide promotes macrophage proliferation and increases tumor cell killing factors (TNF-α, IFN-γ) in vitro. Moreover, T peptide enhances the efficacy of cisplatin and reduces its toxicity in vivo.
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Amygdalin analogues inhibit IFN-γ signalling and reduce the inflammatory response in human epidermal keratinocytes.

Peptide T (PT), an octapeptide fragment located in the V2 region of the HIV-1 gp120-coating protein, appears to be beneficial in the treatment of psoriasis. Our previous investigations suggest that keratinocytes play a key role in conditioning the therapeutic effects of PT in psoriasis. The aim of this study was to explore the effects of PT and the peptidomimetic natural products, Dhurrin and Prunasin, on the expression of the IL-6, IL-8, IL-23, HSP70 and ICAM-1 on IFN-γ and TNF-α-NHEK activated cells. Moreover, we analysed the interference of PT and its analogues through STAT-3 activation. Our results show that the analogues tested exhibit the beneficial biological effects of PT, suggesting the primary role of keratinocytes upon which PT and the peptidomimetics act directly, by reducing proinflammatory responses. Its reduction appears to be important for therapeutic approach in psoriasis pathogenesis.

CC-chemokine ligand 4/macrophage inflammatory protein-1ß participates in the induction of neuropathic pain after peripheral nerve injury.

BACKGROUND: Neuropathic pain is caused by neural damage or dysfunction and neuropathic pain-related symptoms are resistant to conventional analgesics. Neuroinflammation due to the cytokine-chemokine network may play a pivotal role in neuropathic pain. We demonstrate that macrophage inflammatory protein-1ß (MIP-1ß) participates in neuropathic pain. METHODS: Mice received partial sciatic nerve ligation (PSL), and tactile allodynia and thermal hyperalgesia were assessed by von Frey test and Hargreaves test, respectively. Agents were administered into the region surrounding the sciatic nerve (SCN). RESULTS: Using reverse transcription polymerase chain reaction, the mRNA expressions of MIP-1ß and its receptor (CC-chemokine receptor 5; CCR5) in the injured SCN were up-regulated after PSL. MIP-1ß immunoreactivity was localized in macrophages and Schwann cells and increased in the injured SCN on day 1. PSL-induced tactile allodynia on days 4 to 7 was prevented by the administration of MIP-1ß neutralizing antibody (anti-MIP-1ß; on days 0, 3 and 6). PSL-induced up-regulations of inflammatory cytokine-chemokine mRNAs in the injured SCN were suppressed with anti-MIP-1ß treatment on day 7. Administration of CCR5 antagonist, D-ala-peptide T-amide (on days 0, 3 and 6) prevented tactile allodynia and thermal hyperalgesia on days 4 to 14. Single administration of recombinant mouse MIP-1ß (rmMIP-1ß) elicited tactile allodynia. Moreover, rmMIP-1ß increased the mRNA expression of inflammatory mediators in the SCN on day 1 after administration. CONCLUSIONS: These results suggest that MIP-1ß is a novel key mediator, and the peripheral MIP-1ß-CCR5 axis contributes to neuropathic pain. Therefore, investigation of this cascade might be a validated approach for the elucidation of neuropathic pain mechanisms.

Profound anti-HIV-1 activity of DAPTA in monocytes/macrophages and inhibition of CCR5-mediated apoptosis in neuronal cells.

Monocytes/macrophages (M/M) are strategic reservoirs of HIV-1, spreading the virus to other cells and inducing apoptosis in T-lymphocytes, astrocytes and neurons. M/M are commonly infected by R5 HIV-1 strains, which use the chemokine receptor CCR5. D-Ala-peptide T-amide (DAPTA), or Peptide T, named for its high threonine content (ASTTTNYT), is a synthetic peptide comprised of eight amino acids (185-192) of the gp120 V2 region and functions as a viral entry inhibitor by targeting selectively CCR5. The anti-HIV-1 activity of DAPTA was evaluated in M/M infected with R5 HIV-1 strains. DAPTA at 10(-9) M inhibited HIV-1 replication in M/M by > 90%. PCR analysis of viral cDNA in M/M showed that DAPTA blocks HIV entry and in this way prevents HIV-1 infection. Moreover, DAPTA acts as a strong inhibitor and was more active than the non-peptidic CCR5 antagonist TAK-779 in inhibiting apoptosis (mediated by RS HIV-1 strains produced and released by infected M/M) on a neuroblastoma cell line. Our results suggest that antiviral compounds which interfere with receptor mechanisms such as CCR5 could be important, either alone or in combination with other antiretroviral treatments, in preventing HIV infection in the central nervous system and the consequential neuronal damage that leads to neuronal AIDS.

Conformation-activity relationship of peptide T and new pseudocyclic hexapeptide analogs.

Peptide T (ASTTTNYT), a segment corresponding to residues 185-192 of gp120, the coat protein of HIV, has several important biological properties in vitro that have stimulated the search for simpler and possibly more active analogs. We have previously shown that pseudocyclic hexapeptide analogs containing the central residues of peptide T retain considerable chemotactic activity. We have now extended the design of this type of analogs to peptides containing different aromatic residues and/or Ser in lieu of Thr. The complex conformation-activity relationship of these analogs called for a reexamination of the basic conformational tendencies of peptide T itself. Here, we present an exhaustive NMR conformational study of peptide T in different media. Peptide T assumes a gamma-turn in aqueous mixtures of ethylene glycol, a type-IV beta-turn conformation in aqueous mixtures of DMF, and a type-II beta-turn conformation in aqueous mixtures of DMSO. The preferred conformations for the analogs were derived from modeling, starting from the preferred conformations of peptide T. The best models derived from the gamma-turn conformation of peptide T are those of peptides XII (DSNYSR), XIII (ETNYTK) and XVI (ESNYSR). The best models derived from the type-IV beta-turn conformation of peptide T are those of peptides XIV (KTTNYE) and XV (DSSNYR). No low-energy models could be derived starting from the type-II beta-turn conformation of peptide T. The analogs with the most favored conformations are also the most active in the chemotactic test.

Immunomodulatory effects of a set of amygdalin analogues on human keratinocyte cells.

Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. Our previous investigations suggest that keratinocytes play an important role in conditioning the therapeutic effects of the PT in psoriasis. However, peptides are not good therapeutic agents, because they exhibit poor absorption, are easily metabolized and are immunogenic. Using computational methods, the natural product amygdalin was identified as peptidomimetic of PT. However, amygdalin exhibits a toxic profile due to its cyanide group. To overcome this deleterious effect, we synthesized analogues lacking the cyanide group. Human keratinocytes were treated with PT or with three different peptidomimetics of PT. To study its effects on the expression of HSP-70, TGF-beta, alpha-v integrin, ICAM-1 and cytokines, we analysed the protein levels by Western blot and ELISA. Our results show that the different peptidomimetics of PT tested exhibit a similar biological behaviour in regard to the overexpression of HSP-70, TGF-beta and alpha-v integrin than the native peptide. TNF-alpha is overexpressed by PT and SVT-03018; between the other two analogs, SVT-03016 do not produce any significant change in regard to the control, while SVT-03017 shows only a moderate increase in regard to control. SVT-03018 provokes a remarkable upregulation of IL-10, stronger than SVT-03016, SVT-03017 and PT. All the other three analogues reduce comparably to the PT, the expression of ICAM-1 and do not increase the release of proinflammatory cytokines. The results highlighted that the three analogues of amygdalin with the cyanide group removed exhibit the same biological effects of PT. Therefore, they can be considered peptidomimetics, suggesting their possible use in the treatment of psoriasis.

Chemokine receptor-5 (CCR5) is a receptor for the HIV entry inhibitor peptide T (DAPTA).

The chemokine receptor CCR5 plays a crucial role in transmission of HIV isolates, which predominate in the early and middle stages of infection, as well as those, which populate the brain and cause neuro-AIDS. CCR5 is therefore an attractive therapeutic target for design of entry inhibitors. Specific rapid filtration binding assays have been useful for almost 30 years both for drug discovery and understanding molecular mechanisms of drug action. Reported in 1986, prior to discovery of chemokine co-receptors and so thought to act at CD4, peptide T (DAPTA) appears to greatly reduce cellular viral reservoirs in both HAART experienced and treatment naïve patients, without toxicities. We here report that DAPTA potently inhibits specific CD4-dependent binding of gp120 Bal (IC50=0.06 nM) and CM235 (IC50=0.32 nM) to CCR5. In co-immunoprecipitation studies, DAPTA (1 nM) blocks formation of the gp120/sCD4 complex with CCR5. Confocal microscopic studies of direct FITC-DAPTA binding to CCR5+, but not CCR5-, cells show that CCR5 is a DAPTA receptor. The capability of DAPTA to potently block gp120-CD4 binding to the major co-receptor CCR5 explains its molecular and therapeutic mechanism of action as a selective antiviral entry inhibitor for R5 tropic HIV-1 isolates.

Antiviral and immunological benefits in HIV patients receiving intranasal peptide T (DAPTA).

D-Ala-Peptide T-amide (DAPTA), the first viral entry inhibitor, blocks chemokine (CCR5) receptors, not CD4. Early investigators could not "replicate" DAPTAs potent in vitro antiviral effect using the lab-adapted, X4, peptide T-insensitive strain, IIIB, delaying clinical virological studies. We now report that DAPTA, administered to eleven long-term infected (mean=17 years) patients with stable persistent plasma "virus" for up to 32 weeks did not change this level. Infectious virus could not be isolated from their plasma suggesting HIV RNA was devoid of replicative capacity. Progressively less actual virus (P<0.01) could be isolated from white blood cells (PBMCs). DAPTA flushed the monocyte reservoir to undetectable viral levels in most patients. Five of eleven had a mean CD4 increase of 33%. Immune benefits also included a four-fold increase in gamma-interferon-secreting T-cells (antiviral cytotoxic T-cells) in the absence of drug-related toxicity. All five CD4 responders had increases in antiviral T cells and decreases in infected monocytes, an argument for initiating further studies promptly.

Update on D-ala-peptide T-amide (DAPTA): a viral entry inhibitor that blocks CCR5 chemokine receptors.

Peptide T, named for its high threonine content (ASTTTNYT), was derived by a database search which assumed that a relevant receptor binding epitope within env (gp120) would have sequence homology to a known signaling peptide. Binding of radiolabeled gp120 to brain membranes was displaced by peptide T and three octapeptide analogs (including "DAPTA", Dala1-peptide T-amide, the protease-resistant analog now in Phase II clinical trials) with the same potency that these four octapeptides blocked infectivity of an early passage patient isolate. This 1986 report was controversial due to a number of laboratories' failure to find peptide T antiviral effects; we now know that peptide T is a potent HIV entry inhibitor selectively targeting CCR5 receptors with minimal effects on the X4 tropic lab adapted virus exclusively in use at that time. Early clinical trials, which demonstrated lack of toxicity and focused on neurological and neurocognitive benefits, are reviewed and data from a small ongoing Phase II trial--the first to assess peptide T's antiviral effects--are presented. Studies using infectivity, receptor binding, chemotaxis, and blockade of gp120-induced neurotoxicity in vitro and in vivo are reviewed, discussed and presented here. Peptide T and analogs of its core pentapeptide, present near the V2 stem of numerous gp120 isolates, are potent ligands for CCR5. Clinical data showing peptide T's immunomodulation of plasma cytokine levels and increases in the percentage of IFNgamma secreting CD8+ T cells in patients with HIV disease are presented and suggests additional therapeutic mechanisms via regulation of specific immunity.

Immunomodulatory effects of peptide T on human keratinocyte cells.

BACKGROUND: Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. PT is from the V2 region of HIV-1 gp120, an exterior envelope glycoprotein that is a target for host immune responses. The anti-inflammatory mechanisms of PT are not well understood. OBJECTIVES: We studied the immunomodulatory effects of PT on the human keratinocyte cells. METHODS: Cultured human keratinocytes were treated with PT, proteins extracted and analysed by Western blotting and reverse transcriptase polymerase chain reaction. RESULTS: Our findings show reduced expression of intercellular adhesion molecule 1 and an increase in transforming growth factor (TGF)-beta and heat shock protein (HSP)-70 in human keratinocyte cells treated with PT. The HSP-70 increase is modulated by TGF-beta. In fact, we demonstrated that anti-TGF-beta antibodies reduce HSP-70 overexpression. In addition, we show a modulation of alphav integrins after 4 hours of treatment with PT. These receptors favour keratinocyte migration and epidermal regeneration. It has been reported that overexpression of HSP results in dramatic changes to intermediate filaments. These proteins act on keratin intermediate filaments and determine their retraction. The consequence is cell-cell contact detachment and inhibition of cellular hyperproliferation. CONCLUSIONS: Our results support the beneficial effect of PT found in vivo, suggesting, moreover, the primary role of keratinocytes upon which PT acts directly by stimulating the anti-inflammatory function and favouring the regeneration of tissue.

Peptide T bolus normalizes the growth hormone secretion pattern in two children with AIDS.

In humans, HIV infection reduces growth hormone (GH) secretion contributing to AIDS wasting. In rats, the HIV envelope protein gp120 alone blocks GH secretion both in vitro and in vivo through GH-releasing hormone receptors. Peptide T, a modified octapeptide derived from gp120, normalizes GH secretion. We now report that an intravenous bolus of peptide T normalizes nocturnal GH secretion in two out of three children with AIDS. These results, coupled with the lack of toxicity of this experimental AIDS therapeutic, justify clinical trials for AIDS wasting and pediatric AIDS. A clinical and basic science update on peptide T appears in Current HIV Research.

Peptide T revisited: conformational mimicry of epitopes of anti-HIV proteins.

Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations. albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif.

Peptide T-araC conjugates: solid-phase synthesis and biological activity of N4-(acylpeptidyl)-araC.

Due to the capability of peptidyl derivatives of araC to behave as prodrugs of this antimetabolite, and because of the well known biological properties of peptide T and its analogues (in particular that of targeting CD4+ cells), new peptide T-araC conjugates were prepared and tested in vitro for antiproliferative activity. The aim was that of specifically delivering the antitumor drug to CD4+ cells. N4-(Acylpeptidyl)-derivatives of araC were synthesized by a new general approach involving solid-phase synthesis, which allows mild conditions, avoids the usually required protection of the glycoside portion of nucleosides and affords high yields of the final products. After the demonstration that peptide T-araC conjugates were able to activate chemotaxis by binding CD4 receptor on monocyte membranes, the antiproliferative activity was evaluated against a panel of leukemia lymphoma and carcinoma cell lines derived from human tumors, three CD4+ cell lines included. Title compounds resulted effective as antiproliferative agents at concentrations 4- to 10-fold higher than those of araC alone, did not preferentially inhibit CD4+ cells and proved stable not only in cell culture medium containing 20% FCS, but also in human plasma. All this suggests their potential utility in vivo.

Immunomodulatory effects of peptide T on Th 1/Th 2 cytokines.

Peptide T is an octapeptide from the V2 region of HIV-1 gp120. It has been shown to resolve psoriatic lesions--an inflammatory skin disease. The mechanisms of anti-inflammatory actions of peptide T are not well understood. Th1 cytokines such as IL-2, and IFN-gamma are upregulated in psoriasis. These cytokines play a key role in the inflammatory and proliferative processes of psoriasis. The effects of peptide T on Th1 and Th2 cytokines were studied in order to elucidate the mechanisms of antiinflammatory actions of peptide T. It was observed that peptide T at 10(-8) M induces IL-10 production by the human Th2 cell line and PBMC (P < 0.05, ANOVA). Also peptide T at 10(-9) M concentration significantly inhibited IFN-gamma production by PBMC (P < 0.001, ANOVA). Anti IL-10 antibody inhibited the anti-IFN-gamma effect of peptide T (P < 0.05, t-test). Our study shows that peptide T induces IL-10 production and inhibits IFN-gamma production. IL-10 is a potent anti-inflammatory cytokine. It inhibits IL-2 and IFN-gamma production from the T cells and downregulates the expression of TNF-alpha in the antigen presenting cells. Recently, IL-10 has been shown to resolve psoriatic lesions. The effects of peptide T on IL-10 and IFN-gamma production provides a plausible explanation for its clinical efficacy in psoriasis.

VIP and D-ala-peptide T-amide release chemokines which prevent HIV-1 GP120-induced neuronal death.

Vasoactive intestinal peptide (VIP) and DAPTA (D-ala(1)-peptide T-amide, a gp120-derived octapeptide homologous to VIP) prevent neuronal cell death produced by five variants of HIV-1 (human immunodeficiency virus) envelope protein (gp120). VIP or DAPTA treatment of astrocyte cultures resulted in the release of macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES, beta chemokines known to block gp120 interactions with microglial chemokine receptors. In rat cerebral cortical cultures, gp120-induced neuronal killing was partially or completely prevented by chemokines that stimulate the CXCR4, CCR3 or CCR5 chemokine receptors. Chemokines exhibited marked differences in potency and efficacy in preventing toxicity associated with five gp120 variants (LAV/BRU, CM243, RF, SF2, and MN). RANTES had the broadest and most potent inhibition (IC(50)<3 pM for RF isolate). An octapeptide derived from RANTES also exhibited neuroprotection from gp120 (RF isolate) toxicity (IC(50)=0.3 microM). Treatment with chemokines alone had no detectable effect on neuronal cell number. However, antiserum to MIP-1alpha produced neuronal cell death that was prevented by co-treatment with MIP-1alpha, suggesting that this endogenous chemokine exerts a tonic regulation important to neuronal survival. The neuroprotective action of VIP on gp120 was attenuated by co-treatment with anti-MIP-1alpha. These studies suggest that the neuroprotective action of VIP is linked in part to its release of MIP-1alpha. Furthermore, neuroprotection produced by chemokines is dependent on both the type of chemokine and the variant structure of gp120 and may be relevant to drug strategies for the treatment of AIDS dementia.

Anti-chemotactic activities of peptide-T: a possible mechanism of actions for its therapeutic effects on psoriasis.

Peptide T is an octapeptide (ASTTTNYT) from the V2 region of gpl20 of HIV. D-[Ala]-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-amide (DAPTA) is one of its analogue. DAPTA has been shown to resolve the psoriatic lesions. The mechanisms of action of peptide T for its therapeutic effect is not clearly understood. Lymphomononuclear cells play an important roles in inflammatory disease processes. Intraepidermal collection of lymphocytes is a unique feature of the inflammatory processes of psoriasis. It is believed that chemokine such as RANTES (C-C class) plays an important role for intraepidermal localization of the inflammatory infiltrates in psoriasis. In order to study the mechanisms, we have analyzed the effects of DAPTA on monocyte and lymphocyte chemotaxis. Chemotaxis of cells was measured by using Boyden chamber. DAPTA inhibited significantly the monocyte and lymphocyte chemotactic activity of RANTES (p < 0.005, < 0.001). Antichemotactic activities of peptide T analogue could be a possible explanation for its therapeutic efficacy in psoriasis.

Effects of [D-Ala1] peptide T-NH2 and HIV envelope glycoprotein gp120 on cyclic AMP dependent protein kinases in normal and psoriatic human fibroblasts.

In addition to acquired immunodeficiency syndrome (AIDS), persons infected with human immunodeficiency virus often develop cutaneous manifestations, including severe psoriasis. In previous studies, we have established that psoriatic fibroblasts and erythrocytes obtained from psoriatic patients exhibit decreased levels of cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) activity and of 8-azido-[32P]cAMP binding to the RI and RII regulatory subunits of PKA. Because treatment of patients with peptide T (an octapeptide sequence found in the human immunodeficiency virus envelope glycoprotein gp120) has been observed to result in an improvement in the psoriatic condition, studies were initiated to determine if peptide T and gp120 protein treatment of normal and psoriatic human fibroblasts resulted in any changes in PKA. Exposure of psoriatic fibroblasts to peptide T resulted in a time (4 h to 6 d) and dose [10(-14)-10(-8) M] dependent increase in the levels of 8-azido-[32P]cAMP binding to the RI and RII regulatory subunits of PKA, along with a corresponding increase in PKA activity. Peptide T exhibited a biphasic dose dependent response, with maximal effects on PKA noted at 10(-12)M peptide T. Treatment of normal human fibroblasts with peptide T did not result in any change in PKA levels. Conversely, treatment of normal human fibroblasts for 18 h with gp120 protein [10(-13) M] resulted in a significant decrease in the levels of 8-azido-[32P]cAMP binding to RI and RII and in PKA activity. The presence of peptide T blocked this effect of the gp120 protein. These results indicate that peptide T and gp120 protein may inversely alter the intracellular levels of 8-azido-[32P]cAMP binding to RI and RII, and of PKA activity in susceptible cells. These observed changes in the cyclic AMP-PKA signaling pathway, a biochemical marker for psoriasis, may offer some mechanistic insight into the noted beneficial effects of peptide T treatment, including an improvement in psoriatic lesions.

Hypotensive effects of peptide T in conscious rats.

1. The present study investigated the effects of peptide T on mean arterial blood pressure (MAP) in conscious normotensive Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHR) and two-kidney one-clip (2K1C) hypertensive rats. 2. Peptide T was infused via the left jugular vein at a rate of 1 mg/kg per h in SD, SHR and 2K1C rats and then at doses of 0.1, 0.25, 0.5, 1 and 5 mg/kg per h in SHR, with 0.9% saline as a sham control in SHR and 2K1C. Mean arterial pressure was measured directly before, during and after infusion. 3. Peptide T (1 mg/kg per h) decreased blood pressure in both SHR (P < 0.01) and 2K1C (P < 0.05). In normotensive SD rats the fall in MAP approached statistical significance (P = 0.06). The effect of peptide T was not significantly different in normotensive compared with hypertensive rats. Saline infusion had no effect. The blood pressure lowering effect of peptide T appeared to be dose-dependent in SHR.