Chemokine receptor 5 antagonist D-Ala-peptide T-amide reduces microglia and astrocyte activation within the hippocampus in a neuroinflammatory rat model of Alzheimer's disease.

Chronic neuroinflammation plays a prominent role in the progression of Alzheimer's disease. Reactive microglia and astrocytes are observed within the hippocampus during the early stages of the disease. Epidemiological findings suggest that anti-inflammatory therapies may slow the onset of Alzheimer's disease. Chemokine receptor 5 (CCR5) up-regulation may influence the recruitment and accumulation of glia near senile plaques; activated microglia express CCR5 and reactive astrocytes express chemokines. We have previously shown that neuroinflammation induced by chronic infusion of lipopolysaccharide into the 4th ventricle reproduces many of the behavioral, neurochemical, electrophysiological and neuropathological changes associated with Alzheimer's disease. The current study investigated the ability of D-Ala-peptide T-amide (DAPTA), a chemokine receptor 5 chemokine receptor antagonist of monocyte chemotaxis, to influence the consequences of chronic infusion of lipopolysaccharide. DAPTA (0.01 mg/kg, s.c., for 14 days) dramatically reduced the number of activated microglia and astrocytes, as compared with lipopolysaccharide-infused rats treated with vehicle. DAPTA treatment also reduced the number of immunoreactive cells expressing nuclear factor kappa binding protein, a prominent component of the proinflammatory cytokine signaling pathway. The present study suggests that DAPTA and other CCR5 antagonists may attenuate critical aspects of the neuroinflammation associated with Alzheimer's disease.

Peptide T inhibits HIV-1 infection mediated by the chemokine receptor-5 (CCR5).

Peptide T, which is derived from the V2 region of HIV-1, inhibits replication of R5 and dual-tropic (R5/X4) HIV-1 strains in monocyte-derived macrophages (MDMs), microglia, and primary CD4(+)T cells. Little to no inhibition by peptide T was observed with lab adapted X4 viruses such as IIIB, MN, or NL4-3 propagated in CD4(+) T cells or in the MAGI entry assay. The more clinically relevant R5/X4 early passage patient isolates were inhibited via either the X4 or R5 chemokine receptors, although inhibition was greater with R5 compared to X4 receptors. Virus inhibition ranged from 60 to 99%, depending on the assay, receptor target, viral isolate and amount of added virus. Peak inhibitory effects were detected at concentrations from 10(-12) to 10(-9) M. Peptide T acted to block viral entry as it inhibited in the MAGI cell assay and blocked infection in the luciferase reporter assay using HIV virions pseudotyped with ADA envelope. These results using early passage virus grown in primary cells, together with two different entry reporter assays, show that peptide T selectively inhibits HIV replication using chemokine receptor CCR5 compared to CXC4, explaining past inconsistencies of in vitro antiviral effects.

Peptide T blocks GP120/CCR5 chemokine receptor-mediated chemotaxis.

We previously reported that certain short gp120 V2 region peptides homologous to vasaoactive intestinal peptide (VIP), such as "peptide T," were potent inhibitors of gp120 binding, infectivity, and neurotoxicity. The present study shows that synthetic V2-region-derived peptides have potent intrinsic chemotaxis agonist activity for human monocytes and also act as antagonists of high-affinity (0.1 pM) gp120-mediated monocyte chemotaxis. Selectivity is shown in that peptide T is more potent at suppressing M-tropic than T-tropic gp120 chemotaxis. Peptide T was also able to suppress monocyte chemotaxis to MIP-1beta, a chemokine with selectivity for CCR5 chemokine receptors, while chemotaxis of the more promiscuous ligand RANTES was not inhibited, nor was chemotaxis mediated by SDF-1alpha. In order to determine if peptide T mediated its gp120 antagonistic effects via modulation of CCR5 receptors, RANTES chemotaxis was studied using a CCR5 receptor-transfected HOS cell line. In this case, RANTES chemotaxis was potently inhibited by V2-region-derived short peptides. Peptide T also partially suppressed (125)I-MIP1-beta binding to human monocytes, suggesting action at a subset of MIP1-beta receptors. The V2 region of gp120 thus contains a potent receptor binding domain and synthetic peptides derived from this region modulate CCR5 chemokine receptor chemotactic signaling caused by either gp120 or chemokine ligands. The results have therapeutic implications and may explain recent clinical improvements, in that HIV/gp120 actions at CCR5 receptors, such as occur in the brain or early infection, would be susceptible to peptide T inhibition.

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.

Upregulation of RANTES in psoriatic keratinocytes: a possible pathogenic mechanism for psoriasis.

Intraepidermal collections of neutrophils and lymphocytes are unique features of the inflammatory reaction of psoriasis. Migration of leukocytes from dermis to the epidermis suggests a role for chemotactic agent(s). In recent years, increased levels of chemokines such as IL-8 , GRO-a and MCP-1 have been reported in the keratinocytes of psoriatic tissue. IL-8 and GRO-alpha belong to a subfamily (C x C) class and MCP-1 is a beta chemokine. In this study, we investigated RANTES, which is a beta chemokine (C-C class); RANTES has been found to be associated with various cell-mediated hypersensitive disorders. We obtained eight skin biopsies from chronic psoriatic plaques, and five biopsies each from non-lesional psoriatic skin, lichen planus, eczematous dermatitis and skin from healthy controls. Snap-frozen samples were cut into 7 microm cryosections and stained with 6 mg/ml of monoclonal anti-RANTES mouse IgG (DNAX, Palo Alto, CA). Standard immunohistochemistry techniques were applied. RANTES was detected only in the keratinocytes. The number of keratinocytes in per mm2 of epidermis stained for RANTES were 116.79+/-98.42 in psoriatic tissues compared to 32.00+/-46.05 (p<0.05), 6.39+/-3.59 (p<0.01), 2.64 +/-1.15 (p<0.01) and 3.53+/-5.26 (p<0.01), respectively, in the non-lesional, lichen planus, eczematous lesions and normal skin. This is the first study to report that the keratinocytes of psoriatic tissue express high levels of RANTES compared to the controls. IL-8 and related molecules (C x C class) are predominantly chemotactic for neutrophils and MCP-1 is a strong chemotactic factor for monocytes. In contrast, RANTES is chemotactic for memory T cells and activated naive T cells. Increased amounts of RANTES as reported here provide an explanation for migration of the activated T cells to the epidermis of the psoriatic lesions. In addition, RANTES activates T cells. These results suggest that RANTES may have a significant role in the inflammatory process of psoriasis. Our findings further substantiate a regulatory role for keratinocytes in the inflammatory process of psoriasis.

HIV envelope protein gp120 induces neuropeptide Y receptor-mediated proliferation of vascular smooth muscle cells: relevance to AIDS cardiovascular pathogenesis.

Hyperplasia of vascular smooth muscle cells (VSMCs) occurs during HIV infection, part of a spectrum of HIV-mediated cardiovascular and microvascular pathologies. These changes are not due to direct viral infection but may involve the receptor-mediated action of viral proteins, such as the envelope protein gp120. We sought to identify gp120 receptors which might mediate the vascular smooth muscle cell hyperplasia present in HIV infection. A homology between neuropeptide Y (NPY) and the previously identified receptor-active V2-region of gp120 defined by an octapeptide sequence (Peptide T) related to VIP was noted. Since NPY is mitogenic for VSMCs we therefore determined whether gp120 shares this activity. Rat aortic VSMCs were treated for 24 h with human (h)NPY and gp120 in the presence of 0.5% serum to measure [3H]thymidine incorporation, an index of cell proliferation. NPY increased [3H]thymidine incorporation by 80% after a 24-h treatment in a bimodal fashion, with peak effects at 10(-10) M and 10(-8) M. Gp120 was an even more potent mitogen for VSMCs with peak activity occurring at 10(-12) M. Peptide T was equipotent with gp120, and slightly less efficacious, suggesting that this domain may mediate gp120 effects on VSMCs. When combined, gp120 and NPY acted to antagonize one another, lowering DNA synthesis to basal levels. The profile of pharmacologic inhibition supports a role for NPY receptors since antagonists of Y1 and Y2 subtypes substantially or completely inhibited gp120-mediated VSMC proliferation. This is the first demonstration of the proliferative effects of HIV viral protein gp120 on VSMCs. The effect appears to be mediated via gp120 sequences related to VIP, peptide T, and NPY. These ligands may be competitive inhibitors of binding or gp120 processing. Novel treatments may emerge based upon VIP and NPY receptor antagonists if further work substantiates a role for gp120 in the vascular abnormalities of AIDS.

The psychosomatic network: foundations of mind-body medicine.

Research in the 1980s uncovered ubiquitous neuropeptide-receptor distribution in brain structures associated with emotional processing, and throughout many organ systems. This finding supported neuropeptides as biochemical substrates of emotion, and the neuropeptide-receptor network as a parasynaptic system crossing traditional brain-body boundaries. The medical relevance of these findings was affirmed by psychoneuroimmunology research: neuropeptides help to regulate immunocyte trafficking, there is bidirectional communication between nervous and immune system components, immunocytes produce neuropeptides, and nerve cells produce immune-associated cytokines. In the past decade, the concept of a unified psychosomatic network has been strengthened by animal and human research demonstrating relationships between behavior and neuropeptide-mediated regulation of immune functions. Research on emotional expression or disclosure in healthy human subjects as well as in cancer and HIV-positive patients has shown significant positive correlations with clinically relevant immune functions and/or positive health outcomes. Psychosocial interventions emphasizing emotional expression or active coping have evidenced survival benefits in breast cancer and melanoma. These findings suggest that emotional expression generates balance in the neuropeptide-receptor network and a functional healing system. Emotional expression is also a marker for psychospiritual vitalization, and further research should evaluate links between energy-based models of health and neuropeptide-receptor-based models under the rubric of an informational paradigm.

HIV gp120 inhibits the somatotropic axis: a possible GH-releasing hormone receptor mechanism for the pathogenesis of AIDS wasting.

AIDS is often associated with growth retardation in children and wasting in adults. The dissociated envelope protein of the HIV (HIV-1), gp120, can be found in significant concentrations in the parenchyma and cerebrospinal fluid of brains in infected individuals, even in the earliest stages of HIV-1 disease. On the basis of this and the fact that we observed pentapeptide sequence homology between GH-releasing hormone (GHRH) and the V2 receptor-binding region of gp120, we initiated experiments to determine whether gp120 could affect GH secretion and growth in vivo and/or interact with anterior pituitary GHRH receptors in vitro. Although acute IV administration of gp120 in conscious rats had no effect on plasma GH levels, acute administration of gp120 (400 ng) into the brain significantly suppressed pulsatile GH release over a 6-h period compared with saline-injected controls. Furthermore, the putative gp120 antagonist, Peptide T (DAPTA), prevented the suppression of GH by gp120. In support of these in vivo findings, gp120 also significantly (P < 0.05) suppressed GHRH-stimulated GH release in static cultures of dispersed pituitary cells and from cells undergoing perifusion with the peptides. DAPTA prevented the GH suppression by gp120 in both of the pituitary cell paradigms. Furthermore, chronic administration of gp120 into the third ventricle significantly reduced body weight in juvenile rats, compared with saline-injected controls. Thus, gp120 appears to act both at the hypothalamus and pituitary to suppress GH release, and its action at these two locations is associated with a significant loss in body weight in chronically treated young animals. These findings may suggest a specific mechanism for the pathogenesis of wasting in HIV-1 patients that involves blockade of endogenous GHRH receptors by gp120.

Peptide T prevents NBM lesion-induced cortical atrophy in aged rats.

Because VIP is known to be neurotrophic in vitro, the present study tested whether peptide T (PT), an octapeptide with a pentapeptide sequence homologous to VIP, could prevent nucleus basalis (NBM)-induced degenerative changes in the parietal neocortex of aged rats. Aged (20-21 months old) Sprague-Dawley rats were given bilateral neurotoxic lesions of the NBM, and injected daily with PT (1 mg, IP) or vehicle solution for 5 months. Compared to unoperated controls, vehicle-treated NBM lesioned animals had: 1) a significant 17% decrease in overall cortical thickness, 2) significant decreases of 13-29% in the thickness of cortical layers II-IV, V, and VI, and 3) significant neuronal and glial cell loss in layer V. PT treatment prevented or attenuated these lesion-induced decreases in cortical thickness and attenuated the accompanying loss of large neurons in layer V. These results provide evidence that PT1 perhaps acting via VIP receptor stimulation, is neurotrophic and important for the integrity of brain tissue following denervation.

Potent gp120-like neurotoxic activity in the cerebrospinal fluid of HIV-infected individuals is blocked by peptide T.

The envelope protein of the human immunodeficiency virus (gp120) causes neuronal death in developing murine hippocampal cultures or rat retinal ganglion cells. In HIV-infected individuals, gp120 released from HIV-infected macrophages or other cells in the brain has been proposed as the etiology for the pathophysiology of AIDS central nervous system (CNS) disease by diffusing to act at a distance to cause damage and/or death to neighboring neurons. In this study, 28 cerebrospinal fluid (CSF) samples from HIV-infected individuals (79% were WR stage 1 and 2) and neurological disease controls were tested, blind to the investigator, for the presence of in vitro neuronal killing activity. Neurotoxic activity was detected with peak effects at a 1:10(5) dilution in CSF from 9/18 HIV-infected individuals and 1/10 neurological disease controls. Thus half of CSF from early stages of HIV disease are characterized by the presence of neurotoxic activity which is not present in control CSF (Fischers exact test, P < 0.05). The neuronal toxicity by patient CSF could be prevented by peptide T (1 nM). A monoclonal antibody to mouse CD4, RL.172, also attenuated or prevented CSF-induced neuronal killing in all four CSF samples tested. In addition, an antiserum to peptide T previously shown to bind gp120 and neutralize both infectively and direct gp120 neurotoxicity, neutralized the CSF factor. gp120, or a modified small fragment, is suggested to be the responsible toxic molecular entity. These results may be relevant to the pathophysiology of HIV-related CNS disease and the mechanism by which peptide T causes improvements.

Visualization of cholecystokinin receptors on a subset of human monocytes and in rat spleen.

Direct radioreceptor binding experiments and Scatchard analysis reveal CCK receptors on elutriator purified human peripheral blood monocytes, but not on purified human T cells. The monocyte receptors have a single class of high (0.1 nM) affinity binding sites. A structure-function analysis of monocyte binding by different CCK analogs correlates well with previously demonstrated chemotactic responses in monocytes and receptors in brain tissue. Biochemical cross-linking indicates that the monocyte CCK recognition molecule is comparable in molecular size to that in brain membranes. Utilizing a novel fluoresceinated Texas Red-CCK conjugate we have visualized that up to 20% of human peripheral monocytes bear receptors for CCK. A discrete and anatomically significant distribution of CCK receptors in rat spleen is shown by film autoradiography of tissue sections. A more detailed microscopic analysis identifies a dendritic population of monocyte-derived cells within the periarteriolar lymphocyte sheath (PALS) of the white pulp as the CCK receptor-bearing cell in spleen. The anatomical localization of receptor-bearing cells within the PALS region suggests a role for CCK in the antigen processing and sensitization phases of the immune response via regulatory effects of this peptide on a specific, local macrophage-related cell population.

Pharmacokinetics of peptide T in patients with acquired immunodeficiency syndrome (AIDS).

1. The pharmacokinetics of Dala1-peptide T-NH2 (peptide T) was determined during phase I clinical trials in patients with acquired immunodeficiency disease (AIDS) and AIDS related complex (ARC). Drug levels were determined by specific RIA, and in some cases with HPLC analysis, after intravenous (i.v.) or intranasal (i.n.), via metered sprayer, administration. 2. The plasma kinetics appeared to be bi-phasic with a first compartment half-life of 30 to 60 minutes and a second plasma clearance rate of 4 to 6 hours, observed for both routes of administration. Peptide T, in one individual was confirmed to be present at 6 hrs in plasma, determined after HPLC isolation followed by specific RIA. 3. Bioavailability, determined for a 2 mg test dose in six individuals was 9.3 +/- 6.9 nmol/L. Peak plasma levels of 41 +/- 30 nmol/L after 10 mg i.n., 2.8 +/- 5.9 nmol/L after 2 mg i.n., and 0.13 +/- 0.07 nmol/L after 0.4 mg i.n. were observed. In two individuals tested, peptide T was detected in CSF at levels 20% of the corresponding plasma level 90 and 145 minutes post i.v. administration. Peptide T was not detected in urine. I.N. administration was well tolerated for times up to 21 months.

Isolation and identification of a peptide from rat brain which inhibits [3H]TCP binding.

1. A stereospecific radioreceptor binding assay for the phencyclidine analogue [3H]TCP was utilized to screen for inhibitors of binding in extracts of rat brain. 2. Extracts were prepared from rat cortex and hippocampus by methods employing aqueous acid or acidified methanol. Samples were fractionated by reversed phase-HPLC (RP-HPLC) and tested for activity in the radioreceptor assay. Three zones of activity were detected. The most active fraction was further purified by high performance-size exclusion chromatography. 3. Size exclusion chromatography revealed two zones of activity, corresponding to mol. wts of 4000-8000 Da and 1000-2000 Da. Final purification of the lower molecular weight material was achieved by RP-HPLC. 4. Two well-separated peaks were shown to be homogeneous. Their amino acid sequences were determined by automated Edman degradation and data base searching identified these two peaks as the undecapeptide Substance P and its oxidized counterpart (Substance P sulfoxide). 5. Comparative HPLC of synthetic Substance P, or its sulfoxide, as well as spectral analysis confirmed the identity of the isolated peptides. 6. Synthetic Substance P inhibits specific [3H]TCP binding in the radioreceptor assay.

Identification of a domain in cytochrome C that displaces [3H]TCP binding from rat brain membrane receptors: synthesis of beta-neuroprotectin.

1. A stereospecific radioreceptor binding assay for the phencyclidine analogue, [3H]TCP, was utilized to screen for inhibition of binding in extracts of rat brain. 2. Extracts were prepared from rat cerebral cortex and hippocampus by methods employing aqueous acid. The extracts were fractionated by reversed phase-HPLC (RP-HPLC) and tested for activity in the radioreceptor assay. Three zones of activity were detected. The middle zone was further purified by high performance-size exclusion chromatography (HP-SEC). 3. Size exclusion chromatography revealed a single zone of activity corresponding to mol. wts of ca 12,000-31,000 daltons. A fraction from this zone was digested with trypsin, and the resulting enzyme fragments, isolated by a combination of HP-SEC and RR-HPLC, were identified as fragments of rat cytochrome C. 4. Horse cytochrome C was digested with trypsin and the fragments were similarly purified on the basis of the [3H]TCP binding displacement assay. The fragments were sequenced and found to be trypsin cleavage products of a single largely invariant domain of the cytochrome C molecule: Lys-Lys-Lys-Asp-Glu-Arg-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-Lys-Lys. 5. beta-neuroprotectin (D)-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-NH2, inhibits [3H]TCP binding and provides protection against NMDA mediated neuronal cell death at low concentrations.

Influenza viruses induce autoantibodies to a brain-specific 37-kDa protein in rabbit.

Immunization of rabbits with certain H1N1 influenza viruses, including the neurotropic strains NWS/33 and WSN/33 and the New Jersey/76 strain, resulted in the production of autoantibodies to a brain-specific protein of 37 kDa that is present in various species, including humans. Autoantibodies were produced to brain only; various other tissues tested were negative. These antibodies were not elicited by other influenza A or B viruses, including closely related recombinant strains, but were elicited by the isolated hemagglutinin of A/Bellamy/42 strain and by formaldehyde-fixed WSN virus--demonstrating that infection was not essential for the induction of autoantibodies. In histological studies, reaction with anti-viral antisera was specific to gray matter and was confined to sera that recognized the 37-kDa protein. Antibody binding was prominent in regions comprised of neuronal cell bodies in cellular layers of the dentate gyrus, hippocampus, cerebral cortex, and cerebellum and was undetectable in myelin-rich regions, such as the corpus callosum. The 37-kDa protein, therefore, appears to be a neuronal antigen. Antibodies directed against this protein may be involved in the pathogenesis of one or more of the neuropsychiatric disorders that occur after infection with influenza.

Bombesin-like peptides: neuropeptides with mitogenic activity.

In recent years, a remarkable advance has been made in identifying the extracellular factors that control cell proliferation in a variety of cells. Bombesin-like peptides (BN-LP) are neuropeptides involved in the regulation of many important functions, including sensory transmission, regulation of central autonomic pathways, thermoregulation, pituitary, gastric, and pancreatic secretion, food intake, and satiety. They also may stimulate cellular proliferation in a developmental and tissue-specific manner. Their role in pathogenesis appears to be related to their properties as growth factors, especially in the lung, where BN-LP are proposed to induce growth of normal and neoplastic epithelial cells. The formulated hypothesis of control of SCLC growth by BN-LP will be tested using specific synthetic BN antagonists. Neuronal modulation of the release of BN-LP from neuroepithelial bodies and paracrine effects of BN-LP as sensory neurotransmitters indicate possible pathways of nervous system involvement in tissue development, proliferation, and differentiation, as well as repair processes and wound healing.

Neuromodulation by polypeptide growth factors: preliminary results on the distribution of epidermal growth factor receptors in adult brain.

Polypeptide growth factors and their receptors are expressed within the brain and neuromodulatory functions are described. Using autoradiography of 125I-epidermal growth factor (EGF) and radioimmunohistochemistry with an antibody to the EGF receptor, we have determined the distribution pattern of the EGF receptor in adult rat brain. The patterns achieved by both methods were similar at every level of the neuroaxis examined. Highest densities of autoradiographic grains were present in the cingulate cortex, cerebral cortex, hippocampus, amygdala and caudate putamen. This pattern of distribution suggests that EGF may have neuromodulatory functions. Common expression of polypeptide receptors by cells of different organ systems constitutes a biochemical basis for homeostatic network regulation.