Experimental axonopathy induced by immunization with campylobacter jejuni lipopolysaccharide from a patient with guillain‐barré syndrome

Campylobacter jejuni (C. jejunj) infection is the most common antecedent in the axonal variant of Guillain‐Barré syndrome (GBS). Antibodies against nerve gangliosides found in GBS patients recognize cross‐reactive epitopes in the lipopolysaccharide (LPS) of C. jejuni. This led to the molecular mimicry hypothesis of GBS. We immunized eleven rabbits with a LPS extracted from HS:19 C. jejuni strain isolated from a patient with GBS and complete Freund's adjuvant (CFA)(group I). In a second experiment we immunized seven rabbits with LPS, CFA and keyhole limpet hemocyanin (KLH)(group II). All group I rabbits developed high titers of anti‐LPS, anti‐GM1, anti‐GD1b antibodies and lower titers of anti‐GD1a. One rabbit, 50 days after initial inoculation, showed tremor and weakness. All rabbits of group II developed high titres of antiganglioside antibodies and six animals showed weakness 59–113 days after initial inoculation. Two rabbits died. Pathology showed mild to moderate, tendentially grouped, axonal degeneration in sciatic nerves of four out of five animals. Control rabbits of group I (immunized with CFA only) did not develop antibodies, controls of group II (immunized with CFA + KLH) developed low titers of IgG anti‐GM1. None developed neurological signs or showed axonal degeneration. C. jejuni LPS is a potent B‐cell stimulator capable to induce a strong antiganglioside response in rabbits. However, to induce the neuropathy is crucial to employ KLH, a glycoprotein known to stimulate both humoral and cellular responses. This animal model reproduces the pathogenetic process hypothesized in axonal GBS with antiganglioside antibodies post C. jejuni infection.     

Study of experimental cataract produced by sugar alcohols in the organ-cultured mammalian ocular lens

Freshly isolated rabbit lenses were cultured in isosmolar TC-199 medium or hyperosmolar medium containing 180 mM sorbitol or mannitol. These experiments were performed to investigate the probable effects of hyperosmolar media on lens clarity and the ability of lens fiber cells to synthesize membrane intrinsic protein, MP-26. The data from these experiments show that incubation in hyperosmolar medium causes depressed MP-26 synthesis, whereas the presence of sugar alcohols in the culture medium induced anterior and posterior subcapsular opacities. The cation levels of lenses incubated in iso- and hyperosmolar medium were also measured. Data from these experiments revealed that although the experimental lenses display prominent opacities, their cation levels are generally similar to those of control lenses. It is proposed that the observed lens opacities are due to the presence of sugar alcohols in the culture medium and not to hyperosmolar shock.     

Chemical ecology: a view from the pharmaceutical industry.

Biological diversity reflects an underlying molecular diversity. The molecules found in nature may be regarded as solutions to challenges that have been confronted and overcome during molecular evolution. As our understanding of these solutions deepens, the efficiency with which we can discover and/or design new treatments for human disease grows. Nature assists our drug discovery efforts in a variety of ways. Some compounds synthesized by microorganisms and plants are used directly as drugs. Human genetic variations that predispose to (or protect against) certain diseases may point to important drug targets. Organisms that manipulate molecules within us to their benefit also may help us to recognize key biochemical control points. Drug design efforts are expedited by knowledge of the biochemistry of a target. To supplement this knowledge, we screen compounds from sources selected to maximize molecular diversity. Organisms known to manipulate biochemical pathways of other organisms can be sources of particular interest. By using high throughput assays, pharmaceutical companies can rapidly scan the contents of tens of thousands of extracts of microorganisms, plants, and insects. A screen may be designed to search for compounds that affect the activity of an individual targeted human receptor, enzyme, or ion channel, or the screen might be designed to capture compounds that affect any step in a targeted metabolic or biochemical signaling pathway. While a natural product discovered by such a screen will itself only rarely become a drug (its potency, selectivity, bioavailability, and/or stability may be inadequate), it may suggest a type of structure that would interact with the target, serving as a point of departure for a medicinal chemistry effort--i.e., it may be a "lead." It is still beyond our capability to design, routinely, such lead structures, based simply upon knowledge of the structure of our target. However, if a drug discovery target contains regions of structure homologous to that in other proteins, structures known to interact with those proteins may prove useful as leads for a medicinal chemistry effort. The specificity of a lead for a target may be optimized by directing structural variation to specificity-determining sites and away from those sites required for interaction with conserved features of the targeted protein structure. Strategies that facilitate recognition and exploration of sites at which variation is most likely to generate a novel function increase the efficiency with which useful molecules can be created.     

Primary carcinoma of the cystic duct: a case report and review of the literature

The Authors, after a short introduction concerning the primary carcinoma of the cystic duct and the exact definition according to Farrar's criteria, report a case occurred to their observation, the 35th case of international literature. In particular the importance of some hemato-clinical parameters and instrumental investigation (ERCP, angio-CT) to underlined in order to surgical indication. In the case here reported cholecystectomy uses informed with partial resection of the hepato-choledochus and excision of some periductal and pericholedochus lymph nodes. Finally, the Authors discuss about clinical data and diagnostic and therapeutic trends, on the case of their experience and literature review.     

VEGF inhibition and cytotoxic effect of aplidin in leukemia cell lines and cells from acute myeloid leukemia.

BACKGROUND: Aplidine (APL) is a marine depsipeptide isolated from the Mediterranean tunicate Aplidium albicans that is under clinical phase II development. In contrast to the lack of bone marrow toxicity reported in phase I/II studies, it has been shown to induce cytotoxicity at very low concentration against lymphoblastic leukemia blast, as well as having an impact in the vascular endothelial growth factor (VEGF)/VEGF receptor 1 loop. PATIENTS AND METHODS: To confirm these findings we investigated APL-related VEGF inhibition and its cytotoxic effect on myeloid leukemic cells lines (K-562, HEL and HL60) and fresh leukemia blasts derived from 30 patients with acute myeloid leukemia (AML). The conventional active 4-demetoxi-daunorubicin (idarubicin; IDA) was included as a positive control. RESULTS: APL was found to be significantly (P<0.001) more active than IDA in obtaining 50% growth-inhibition in K-562, HEL and HL60 cell lines. Results obtained with AML blast cells were super imposible. ID(50) ranged from 0.024 to 0.610 microM for IDA (0.200+/-0.176) and from 0.001 to 0.108 microM for APL (0.020+/-0.031). Annexin V tests and cell cycle analysis performed on cell lines confirmed the stronger citotoxic capability of APL as apoptotic inducer and as a G(1) blocker. The inhibitory effects of APL on VEGF release and secretion have been confirmed by ELISA tests performed on HEL: the VEGF concentration in cell surnatant was reduced from 169 to 36 pg/ml after 24 h of exposure to a pharmacological concentration of APL. CONCLUSIONS: APL harbors a strong in vitro antileukemic activity at a concentration achievable in patients at non-myelotoxic doses. Our data also support the notion of an impact on VEGF secretion. Clinical studies with this new marine-derived compound in relapsed/resistant leukemia are underway.     

Favorable toxicity profile of raltitrexed in elderly patients treated for colorectal cancer: a case series

BACKGROUND: Age-related physiological changes may lead to an increased toxicity of chemotherapy in the elderly, thus making tumor treatment difficult in this increasing subset of patients. OBJECTIVE: Since many trials claimed a favorable therapeutic index with raltitrexed, the aim of our preliminary study was to evaluate the anticancer activity and the toxic profile of this drug in the elderly. METHODS: Thirteen elderly patients with colorectal cancer, aged 75-90 years, were enrolled in a monochemotherapy treatment with raltitrexed. Due to their advanced age, the drug was administered with a 33% reduction of the dose. RESULTS: One partial response, four disease stabilizations, and two disease progressions were observed in 7 patients with advanced colorectal cancer. The patients with response or disease stabilisation had a satisfactory time to progression. Four out of 6 patients treated in the adjuvant setting for Dukes' C colorectal cancer remain disease free at observation periods of 15+ to 29+ months. Toxicity was virtually absent in all patients. CONCLUSIONS: The activity of monochemotherapy with raltitrexed appears to be appealing, above all because it is observed in the absence of toxicity. Though recent reports suggest some concern about severe complications of treatment with raltitrexed, administration of reduced doses of this drug seems to be a putative therapy for those patients who, because of their age, are highly susceptible to the adverse effects of chemotherapy.     

Long-term suppression of EAE relapses by pharmacological impairment of epitope spreading

Background and PurposeImmune events sustaining dendritic cell (DC)-dependent epitope spreading (ES) are of key relevance to the development of relapses during multiple sclerosis (MS). Although no drugs are currently available to target ES, its inhibition would represent a major advancement in MS therapy. Inhibitors of the enzyme PARP-1 afford protection in animal models of MS, such as experimental autoimmune encephalomyelitis (EAE). These drugs epigenetically impair antigen presentation by DCs, but whether these drugs affect ES is unknown. Here, we investigated whether short-term treatments with these compounds would impair ES, thereby preventing EAE relapses.Experimental ApproachWe used a model of relapsing EAE in SJL mice and also adopted in vivo and ex vivo models of DC-dependent T-cell polarization. The effect of PARP-1 inhibitors on ES was evaluated at the humoral and cellular level.Key ResultsShort-term treatments with PARP-1 inhibitors during the acute phase of relapsing EAE of mice induced, at later times, more tolerogenic DCs, increased numbers of Treg cells and impairment of ES at the humoral and cellular level. These effects are followed by long-lasting reduction of relapse severity and incidence, although drug treatment had been discontinued for several weeks. PARP-1 inhibitors also induced tolerogenic DCs and increased Treg cells number and function in a model of ovalbumin immunization.Conclusions and ImplicationsOur data emphasize the therapeutic potential of PARP-1 inhibitors in the treatment of relapsing-remitting MS and additional ES-driven autoimmune disorders.     

Immunoprecipitation of the parathyroid hormone receptor.

An 125I-labeled synthetic analog of bovine parathyroid hormone, [8-norleucine,18-norleucine,34-tyrosine]PTH-(1-34) amide ([Nle]PTH-(1-34)-NH2), purified by high-pressure liquid chromatography (HPLC), was employed to label the parathyroid hormone (PTH) receptor in cell lines derived from PTH target tissues: the ROS 17/2.8 rat osteosarcoma of bone and the CV1 and COS monkey kidney lines. After incubation of the radioligand with intact cultured cells, the hormone was covalently attached to receptors by using either a photoaffinity technique or chemical (affinity) cross-linking. In each case, covalent labeling was specific, as evidenced by a reduction of labeling when excess competing nonradioactive ligand was present. After covalent attachment of radioligand, membranes were prepared from the cells and solubilized in the nonionic detergent Nonidet P-40 or octyl glucoside. The soluble membrane fraction present in the supernatant of a 100,000 X g centrifugation was incubated with IgG prepared from anti-PTH antiserum generated to the amino-terminal region, residues 1-34, of PTH. The IgG-PTH-receptor complex was precipitated with staphylococcal protein A-Sepharose. Analysis of the immunoprecipitate on NaDod-SO4/polyacrylamide gel electrophoresis followed by autoradiography revealed the presence of a doublet of apparent molecular mass 69-70 kDa. Specifically labeled bands of approximate molecular mass 95 and 28 kDa were also observed. The anti-PTH IgG was affinity purified by passage over a PTH-Sepharose column and used to make an immunoaffinity column. The 70- and 28-kDa bands were also observed after labeled solubilized membrane preparations were allowed to bind to this column and then were eluted by using a [Nle]PTH-(1-34)-NH2-containing buffer or acetic acid. These studies suggest that the use of an anti-PTH antiserum that binds receptor-bound hormone is likely to be a useful step in the further physiochemical characterization and purification of the PTH receptor.