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A synthetic 17-amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins has been found to exhibit suppressive properties for numerous immune functions. It has been shown that CKS-17 causes an imbalance of human types 1 and 2 cytokines and inhibition of the immune responses of lymphocytes, monocytes, and macrophages. CKS-17 induced increased intracellular levels of cAMP, which plays an important role in regulation of cytokine biosynthesis. In this study, using a Jurkat T-cell line and Western blot analysis, CKS-17 induced phosphorylation of PLC-gamma1, Raf-1, MEK and ERK1/2. Using a PLC selective inhibitor U73122 or PLC-gamma1-deficient Jurkat cell line, phosphorylation induced by CKS-17 of ERK1/2, PLC-gamma1, or Raf-1, respectively, were undetectable or significantly reduced. Reintroduction of PLC-gamma1 into the PLC-gamma1-deficient Jurkat cells restored the phosphorylation of ERK1/2 and PLC-gamma1 induced by CKS-17. Further, pretreatment of Jurkat cells with PKC inhibitors blocks the phosphorylation of Raf-1, MEK, and ERK1/2 induced by CKS-17. These results indicate that CKS-17 induces the PLC-gamma1-PKC-Raf-1-MEK-ERK1/2 signaling pathway.

In vivo tests were performed to assess the influence of the protease inhibitor TL-3 on feline immunodeficiency virus (FIV)-induced central nervous system (CNS) deficits. Twenty cats were divided into four groups of five animals each. Group 1 received no treatment, group 2 received TL-3 only, group 3 received FIV strain PPR (FIV-PPR) only, and group 4 received FIV-PPR and TL-3. Animals were monitored for immunological and virological status, along with measurements of brain stem auditory evoked potential (BAEP) changes. Groups 1 and 2 remained FIV negative, and groups 3 and 4 became virus positive and seroconverted by 3 to 5 weeks postinoculation. No adverse effects were noted with TL-3 only. The average peak viral load for the virus-only group 3 animals was 1.32 x 10(6) RNA copies/ml, compared to 6.9 x 10(4) copies/ml for TL-3-treated group 4 cats. Group 3 (virus-only) cats exhibited marked progressive delays in BAEPs starting at 2 weeks post virus exposure, which is typical of infection with FIV-PPR. In contrast, TL-3-treated cats of group 4 exhibited BAEPs similar to those of control and drug-only cats. At 97 days postinfection, treatments were switched; i.e., group 4 animals were taken off TL-3 and group 3 animals were treated with TL-3. BAEPs in group 3 animals returned to control levels, while BAEPs in group 4 animals remained at control levels. After 70 days on TL-3, group 3 was removed from the drug treatment regimen. Delays in BAEPs immediately increased to levels observed prior to TL-3 treatment. The findings show that early TL-3 treatment can effectively eliminate FIV-induced changes in the CNS. Furthermore, TL-3 can counteract FIV effects on the CNS of infected cats, although continued treatment is required to maintain unimpaired CNS function.

Background: Mannose-binding lectin (MBL), an acute-phase serum protein of hepatic origin, plays an essential protective role in host innate immunity in targeting microbial pathogens for destruction via opsonization, enhancement of phagocytosis and complement activation. MBL deficiency, characterized by low serum MBL, has been attributed to genetic mutations in both structural and promoter regions of the gene coding for the protein. Concomitant MBL deficiency in patients with chronic immunologic disease has been associated with increased susceptibility to complicating infections that may hasten disease progression., Objective: Few cases of inherited MBL deficiency in adults and possible associations with recurrent infection have been reported. To address this issue, we investigated the MBL profile of four generations within a single adult family whose members have experienced a variety of persistent infections., Methods: MBL serum levels and MBL genotypes of each participating family member were ascertained by enzyme-linked immunoadsorbent assay and reverse transcriptase-polymerase chain reaction, respectively. MBL complement activation, as measured by C4b deposition against mannan-coated wells, was assayed using an enzyme-linked immunoadsorbent assay. Routine immunologic and cellular tests were carried out to evaluate the immunologic status of each family member., Results: Six of the 7 family members screened carried one or more of the variant MBL alleles in their genotype and had correspondingly low serum MBL and reduced ability to affect C4b opsonization. Medical histories of the participating family members revealed an array of mild to severe recurrent infections despite no apparent immunodeficiency., Conclusions: Our studies show that MBL deficiency is an inherited characteristic and may be a crucial factor in maintaining immunologic health.

Based on the substrate transition state and our strategy to tackle the problem of drug resistance, a series of HIV/FIV protease (HIV /FIV PR) monocyclic inhibitors incorporating a 15- or 17-membered macrocycle with an equivalent P3 or P3' group and a unique unnatural amino acid, (2R, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid, have been designed and synthesized. In addition, based on the structure of TL3 with small P3/P3' group, we have synthesized two conformationally restricted bicyclic inhibitors containing the macrocycle, which mimic the P1/P1'-P3/P3' tripeptide [Phe-Val-Ala] of TL3. We have found that the contribution of the macrocycle in our monocyclic inhibitors is important to the overall activity, but the ring size does not affect the activity to a significant extent. Several inhibitors that were developed in this work, exhibit low nanomolar inhibitory activity against the wild-type HIV/FIV PR and found to be highly effective against some drug-resistant as well as TL3-resistant mutants of HIV PRs. Compound 15, in particular, is the most effective cyclic inhibitor in hand to inhibit FIV replication in tissue culture at a concentration of 1.0 micro g/mL (1.2 microM).

CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.