Advanced malignant melanoma has a poor prognosis since chemotherapy is mostly ineffective due in part to the intrinsic and/or extrinsic resistance of melanoma cells to systemic treatment with anti-neoplastic agents. The reasons for the chemoresistant phenotype are unknown. The relevance of well-analyzed drug-resistance mechanisms, e.g., intracellular/ extracellular transport and induction of certain enzyme systems, is reviewed. Most anti-cancer drugs kill susceptible cells through induction of apoptosis. Therefore, it appears that differences in the apoptotic pathways which lead to apoptotic deficiency may account for the ability of some tumor cells to resist drug therapy. Human melanomas, which are characteristically drug-resistant, are more likely to have altered apoptotic pathways and fewer pro-apoptotic molecules. Tumor cells with these characteristics are seldom sensitive to drugs. The complexity of the molecular variants involved in signal transduction along apoptotic pathways suggests that the cell may have a variety of possibilities for regulating apoptosis and generating apoptotic deficiency. Thus, apoptosis and apoptotic deficiency should be analyzed to better clarify the mechanisms of melanoma resistance.

Tick-borne relapsing fever in North America is primarily caused by the spirochete Borrelia hermsii. The pathogen employs multiple strategies, including the acquisition of complement regulators and antigenic variation, to escape innate and humoral immunity. In this study we identified in B. hermsii a novel member of the complement regulator-acquiring surface protein (CRASP) family, designated BhCRASP-1, that binds the complement regulators factor H (FH) and FH-related protein 1 (FHR-1) but not FH-like protein 1 (FHL-1). BhCRASP-1 specifically interacts with the short consensus repeat 20 of FH, thereby maintaining FH-associated cofactor activity for factor I-mediated C3b inactivation. Furthermore, ectopic expression of BhCRASP- 1 converted the serum-sensitive Borrelia burgdorferi B313 strain into an intermediate complement-resistant strain. Finally, we report for the first time that BhCRASP-1 binds plasminogen/plasmin in addition to FH via, however, distinct nonoverlapping domains. The fact that surface-bound plasmin retains its proteolytic activity suggest that the dual binding specificity of BhCRASP-1 for FH and plasminogen/plasmin contributes to both the dissemination/invasion of B. hermsii and its resistance to innate immunity.

Anticancer drugs kill susceptible cells through induction of apoptosis. Alterations of apoptotic pathways in drug-resistant tumor cells leading to apoptosis deficiency might represent a potent mechanism conferring drug resistance. We have assessed the effect of etoposide and cisplatin on the apoptotic pathways of the drug-sensitive human melanoma cell line MeWo as well as its etoposide- and cisplatin-resistant sublines (MeWo(Eto01), MeWo(Eto1), (and) MeWoCis01, MeWo(Cis1)). Etoposide and cisplatin induced apoptosis in drug-sensitive MeWo cells as indicated by dose-dependent (i) cytochrome c release, (ii) caspase activation, (iii) DNA fragmentation, and (iv) cleavage of poly(ADP-ribose)polymerase. In contrast, whereas low etoposide-resistant cells (MeWo(Eto01)) demonstrated reduced but detectable apoptotic activities, highly etoposide-resistant cells (MeWo(Eto1)) did not exhibit any of the apoptotic events observed in etoposide-induced cell death downstream of a strongly reduced cytochrome c release. Highly cisplatin-resistant cells (MeWo(Cis1)), however, demonstrated a reduced caspase 9 activity and cytochrome c release but the extent of effector caspase activation as well as DNA fragmentation was comparable to that of sensitive MeWo cells at equitoxic concentrations. In addition, poly(ADP-ribose)polymerase cleavage was strongly reduced in highly cisplatin-resistant sublines. Taken together, sensitive and drug-resistant MeWo cells utilized different apoptotic pathways upon drug exposure in a drug-dependent fashion and apoptosis deficiency was strongly associated with the drug-resistant phenotype.

Serological diagnosis of Lyme disease may be complicated by antigenic differences between infecting organisms and those used as test references. Accordingly, it would be helpful to include antigens whose sequences are well conserved by a broad range of Lyme disease spirochetes. In the present study, line blot analyses were performed using recombinant complement regulator-acquiring surface protein 2 (BbCRASP-2) from Borrelia burgdorferi sensu stricto strain B31 and serum samples from human Lyme disease patients from throughout the United States and Germany. The results indicated that a large proportion of the patients had produced antibodies recognizing recombinant BbCRASP-2. In addition, Lyme disease spirochetes isolated from across North America and Europe were found to contain genes encoding proteins with high degrees of similarity to the B. burgdorferi type strain B31 BbCRASP-2, consistent with the high percentage of serologically positive patients. These data indicate that BbCRASP-2 may be valuable for use in a widely effective serological assay.

The complement regulator-acquiring surface protein (CRASP)-1 is a member of the paralogous gene family gbb54 and the dominant FHL-1 and factor H binding protein of Borrelia burgdorferi sensu stricto (s.s.). It was shown recently that expression of BbCRASP-1 directly correlates with serum resistance of B. burgdorferi s.s. isolates. In the present study we have elucidated the putative potential of other members of the gbb54 paralogous family, including orthologs ZSA66, ZSA69, ZSA70, ZSA71, ZSA72 and ZSA73 of the European B. burgdorferi s.s. strain ZS7, to bind human FHL-1 and factor H. In spite of their overall similarity in protein sequence, between 47% and 67%, and the fact that the C-terminal region of ZSA69 shows 70% similarity with BbCRASP-1, none of the orthologous proteins was able to bind human FHL-1 and/or factor H. BbCRASP-1 is the only member of the paralogous gene family gbb54 that binds to human complement regulators, supporting the notion that BbCRASP-1 plays a critical role in evasion of complement by B. burgdorferi s.s. and thus may be helpful in the development of novel therapeutic strategies against Lyme borreliosis.

Previous studies using small numbers of serum samples from human patients and experimentally infected animals identified the frequent presence of antibodies recognizing RevA, a borrelial fibronectinbinding outer surface protein. We now demonstrate that most examined Lyme disease spirochetes from North America and Europe contain genes encoding RevA proteins, some with extensive regions of conservation and others with moderate diversity. Line blot analyses using recombinant RevA from two diverse Lyme disease spirochetes of RevA and serum samples from culture-confirmed human Lyme disease patients from the United States (n ‫؍‬ 46, mainly with early Lyme disease) and Germany (>500, with early and late manifestations of Lyme disease) were performed. The results indicated that a sizable proportion of patients produced antibodies that recognized recombinant RevA. Overall, RevA-based serological studies were less sensitive and less specific than other assay types, such as the VlsE-based C6 peptide assay. However, sera from patients in the initial stages of Lyme disease contained antibodies against RevA, demonstrating that this protein is expressed early in human infection. Thus, RevA may be a useful target for preventative or curative therapies.

Lyme disease (LD) is a tick-borne infection caused by the bacterial pathogen Borrelia burgdorferi. Current diagnostic tests mostly use borrelial lysates or select antigens to detect serum antibodies against B. burgdorferi. These immunoassays are not entirely effective, especially for detection of early infection. We have recently characterized an in vivo-induced antigen, BBK07, as a serodiagnostic marker for LD. We now report that in a line blot assay, recombinant BBK07 protein-based detection is 90% sensitive and nearly 100% specific against B. burgdorferi infection in humans. Using an overlapping peptide library of 23 peptides encompassing fulllength BBK07, we identified the immunodominant epitopes of BBK07 during human infection. We show that a select combination of amino-terminal peptides significantly enhanced BBK07-based diagnostic accuracy compared to that with the full-length protein. Although in enzyme-linked immunosorbent assay (ELISA) studies BBK07 peptides had overall lower sensitivity than established serodiagnostic peptides, such as the VlsE peptide C6 and OspC peptide pepC10, for the detection of early human LD, a subset of serum samples that failed to recognize either VlsE or OspC peptides were preferentially reactive to BBK07 peptides. These results highlight the fact that BBK07 peptides could be useful to complement the efficacy of VlsE and OspC peptidebased serodiagnostic assays. Finally, using a panel of canine sera, we show that BBK07 peptide is also effective for LD diagnosis in infected dogs. Together, our data show that peptides from the B. burgdorferi surface protein BBK07 are highly specific and sensitive serodiagnostic markers, and we suggest their future use in LD diagnostic assays.

Borrelia burgdorferi complement regulator-acquiring surface protein 1 (CRASP-1), the dominant factor H and FHL-1-binding protein of the Lyme disease spirochete B. burgdorferi, is implicated in pathogen persistence and was recently reported to be nonimmunogenic in humans. Here we show that serum samples from Lyme disease patients contain antibodies with exclusive specificity for nondenatured structural determinants of CRASP-1.