Alexander M. Abdelnoor

Endotoxin-induced local inflammation and hyperalgesia in rats and mice: a new model for inflammatory pain

&NA; Lipopolysaccharide, also known as endotoxin (ET), is a major constituent of the outer membrane of the cell wall of most gram negative bacteria. ET is known to cause a number of pathophysiological changes associated with illness including inflammatory pain. The aim of this study is to characterize the peripheral hyperalgesia induced by ET in rats and mice. Different groups of rats and mice received different doses of ET ranging from 0.6 &mgr;g to 40 &mgr;g dissolved in 50 &mgr;l saline and injected in the plantar area of the left hind legs. All animals were subjected to tail immersion (TF), hot plate (HP) and paw pressure (PP) tests, 2–3 days prior to ET injection and during the following 1–2 days. ET injections produced a dose‐dependent decrease in the latencies of the HP and PP tests of the injected leg reaching a maximum decrease of 50–60% of the control with 20–40 &mgr;g ET at 9 h (rats) and 24 h (mice) after the injection. Almost complete recovery was observed after 24 h in rats and 48 h in mice. TF latencies showed a less but a significant decrease while PP of the opposite leg and all tests in saline‐injected animals did not elicit significant variations and served as additional controls. Our results indicate that the use of ET‐produced hyperalgesia is a valid model for local and reversible inflammatory pain, with minimal distress to the animal. This model can also be used to study the efficacy of various anti‐inflammatory and analgesic drugs and the molecular mechanisms of inflammation induced by bacterial invasion.

The potential use of toll-like receptor (TLR) agonists and antagonists as prophylactic and/or therapeutic agents

Toll-like receptors (TLR) and their ligands are one of the main players in the initiation of innate immunity which precedes, and is required, for the establishment of adaptive immunity. Manipulating the immune response by using TLR agonists or antagonists might be of therapeutic and/or prophylactic value. This review covers; 1-TLR. their natural ligands and ligand - TLR signaling events, 2-TLR againsts and their use in clinical trials as vaccine adjuvants, and to treat allergy, cancer and infectious diseases, 3-TLR antagonists and their use in clinical trials to treat septic shock and autoimmune diseases. Potential drawbacks related to their potential use as prophylactic and/or therapeutic agents are discussed.

Two potential improvements to BCG and their effect on skin test reactivity in the Lebanon

An account of an ongoing project to assess the possible benefits of two additives to BCG vaccine is presented. These additives are suspensions of irradiation killed Mycobacterium vaccae in one case, and M. leprae in the other. Groups of children aged 7-17 living in Zgharta and Akkar districts of North Lebanon have received vaccination with BCG alone or with either of the two additives since 1980. This region was chosen since contact with environmental mycobacteria is small, but both leprosy and tuberculosis occur there. So far the effects of the additives have been assessed by annual skin testing of volunteers with Tuberculin, Leprosin A, Vaccin and Scrofulin, and by measuring the size of the vaccine scars. Some children have now been followed up on four occasions, and special attention is paid to them. No complications have been encountered in the 1740 children who have entered the study (by our observation, or by local report on those who have not attended for follow-up) and the mean scar sizes after vaccines with the additives are no larger than those after BCG alone. There is no evidence that the additives have prevented development of Tuberculin positivity after vaccination, or have changed the nature of reaction to it. Incorporation of M. leprae significantly increased Leprosin A positivity and both additives increased Vaccin positivity in comparison with the effects of BCG alone. The results are fitted to a model of the theoretical expectations of the study and may be beginning to show the advantages expected of the additives. The only unexpected finding was a reduction in Scrofulin positivity especially associated with the additives. The very low contact with environmental mycobacteria experienced in the study area has allowed the pattern of post-vaccination decay of skin test positivity to be studied with greater precision than has been reported before, and differences have been detected between the two districts where the study was conducted. Confirmation of the possible advantage of the additives will rest with data to be obtained in longer term follow-ups and in studies being carried out in other countries.

Effects of Various Analgesic and Anti-Inflammatory Drugs on Endotoxin-lnduced Hyperalgesia in Rats and Mice

A new model of endotoxin (ET)-induced hyperalgesia has been used to test the effects of four classes of drugs in rats and mice. Hyperalgesia was assessed by paw pressure (PP), hot plate (HP) and tail flick (TF) tests. Each drug was injected intraperitoneally 24 and 12 h before ET injection and just before each pain test at 3, 6, 9 and 24 h after ET injection. At the dosages used, acetaminophen and dexamethasone were the most effective in reducing PP hyperalgesia and least effective on TF hyperalgesia, while indometacin and morphine produced their main effect on TF hyperalgesia. The four drugs were about equally effective in reversing HP hyperalgesia. We conclude that ET hyperalgesia is mediated by both prostaglandin-sensitive and prostaglandin-independent mechanisms.

Skin sensitization to mycobacteria amongst school children prior to a study of BCG vaccination in North Lebanon

1888 school children aged between 7 and 17 years, living in 13 villages in two districts of North Lebanon, were skin tested with four new tuberculins as the initial step in a study of BCG vaccination. The great majority of children were tested with Tuberculin, Leprosin A, Vaccin and Scrofulin. In comparison with other countries where similar studies have been carried out, extremely low levels of sensitization were discovered, indicating very little contact with mycobacteria. There was, however, a statistically significant increase in positivity with increasing age. The results obtained for the villages of each district were significantly different from each other, positivity being greatest in Akkar district for each reagent. The eight villages of Zgharta district could be separated into a lowland group of four villages, a mountain group of three villages and one anomalous mountain village. There was significantly more positivity in the lowland than in the mountain villages. In Akkar district, where leprosy has a low prevalence, positivity to Leprosin A was 8% amongst the children (leaving out an anomalous village). In Zgharta district where the disease does not occur, positivity was 3.4% to Leprosin A for the lowland villages and 0.9% for the group of 3 mountain villages. The two anomalous villages were the only ones in which tuberculosis cases were known to have occurred recently, and they were the only two villages in which Tuberculin positivity exceeded 10%.

Streptococcus sp. and Staphylococcus aureus Isolates from Patients with Psoriasis Possess Genes That Code for Toxins (Superantigens): Clinical and Therapeutic Implications

Superantigens are powerful T lymphocyte–stimulating agents that are believed to contribute to the pathogenesis of certain diseases such as psoriasis. Toxins produced by Streptococcus pyogenes and Staphylococcus aureus are superantigens. The aim of this study was to detect genes that code for superantigens in Streptococcus and Staphylococcus aureus isolates from psoriatic patients. Primers to amplify streptococcal pyrogenic exotoxin A, B, and C and streptolysin O genes and staphylococcal enterotoxin A, B, C, and D genes were used. Streptococcal exotoxin B was detected in five streptococcal isolates. Staphyloccocus aureus enterotoxin A and/or C genes were detected in nine S. aureus isolates. Isolates from 13 of 22 patients possesed gene(s) that code for toxin(s) (superantigens). These results might support the role of superantigens in the exacerbation of psoriasis.

Role of rifampicin in limiting Escherichia coli O157:H7 Shiga-like toxin expression and enhancement of survival of infected BALB/c mice

The sequelae of infection with Escherichia coli O157:H7 include the potentially fatal haemolytic uraemic syndrome. The pathobiological process of E. coli O157:H7 is chiefly dependent on the production of Shiga-like toxins I and II (SLT-I and -II). Antibiotic treatment is currently refrained from since it may lead to enhanced release of SLTs from the bacterium. In this study, the potential utility of rifampicin in treating E. coli O157:H7 infections was assessed both in vitro and in vivo. Five strains of E. coli O157:H7 were tested by reverse transcriptase polymerase chain reaction (RT-PCR) for the transcription of the SLT-I- and SLT-II-encoding genes (stx1 and stx2, respectively). Treatment of bacterial strains with the rifampicin minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), or the MIC followed by the MBC led to the inhibition of stx1 and stx2 gene transcription. Treatment with the MIC or with the MIC followed by the MBC was also capable of limiting toxin release. SLT-I and SLT-II detection by reverse passive latex agglutination showed an effective decrease in toxin titres following treatment with the MIC of rifampicin or with the MIC followed by the MBC. Treatment of cultures with the MBC alone was not as effective in decreasing toxin titres. The efficacy of rifampicin in treating E. coli O157:H7-infected BALB/c mice was also assessed. Rifampicin treatment resulted in enhanced mouse survival and limited the weight loss of infected animals. In conclusion, both in vitro and in vivo tests showed that rifampicin may be useful in treating E. coli O157:H7 infection.

Decrease in Shiga toxin expression using a minimal inhibitory concentration of rifampicin followed by bactericidal gentamicin treatment enhances survival of Escherichia coli

BackgroundTreatment of Escherichia coli O157:H7 infections with antimicrobial agents is controversial due to an association with potentially fatal sequelae. The production of Shiga toxins is believed to be central to the pathogenesis of this organism. Therefore, decreasing the expression of these toxins prior to bacterial eradication may provide a safer course of therapy.MethodsThe utility of decreasing Shiga toxin gene expression in E. coli O157:H7 with rifampicin prior to bacterial eradication with gentamicin was evaluated in vitro using real-time reverse-transcription polymerase chain reaction. Toxin release from treated bacterial cells was assayed for with reverse passive latex agglutination. The effect of this treatment on the survival of E. coli O157:H7-infected BALB/c mice was also monitored.ResultsTranscription of Shiga toxin-encoding genes was considerably decreased as an effect of treating E. coli O157:H7 in vitro with the minimum inhibitory concentration (MIC) of rifampicin followed by the minimum bactericidal concentration (MBC) of gentamicin (> 99% decrease) compared to treatment with gentamicin alone (50-75% decrease). The release of Shiga toxins from E. coli O157:H7 incubated with the MIC of rifampicin followed by addition of the MBC of gentamicin was decreased as well. On the other hand, the highest survival rate in BALB/c mice infected with E. coli O157:H7 was observed in those treated with the in vivo MIC equivalent dose of rifampicin followed by the in vivo MBC equivalent dose of gentamicin compared to mice treated with gentamicin or rifampicin alone.ConclusionsThe use of non-lethal expression-inhibitory doses of antimicrobial agents prior to bactericidal ones in treating E. coli O157:H7 infection is effective and may be potentially useful in human infections with this agent in addition to other Shiga toxin producing E. coli strains.

Plasmid DNA Vaccines

Genes that code for the production of protein antigens have been cloned and recombined with plasmids. Gene-plasmid constructs have been amplified in a bacterial host, purified and administered to a mammalian host. The gene is expressed in the host and the antigen that is produced induces an immune response. These so-called DNA vaccines have been prepared for a number of infectious agents, some tumors and some allergens, and were shown to be efficacious in animal studies. Clinical trials for some of these vaccines are underway. Advantages of using a DNA vaccine include the abilities to favor a T helper-1 or a T helper-2 lymphocyte response and to induce a cytotoxic T-lymphocyte response. Moreover, some reports have indicated that they produce long-lasting immunity. DNA vaccines might be used in situations where no effective vaccine is available for a disease. However, their use might not be risk-free. Further research in this field is needed to determine their efficacy and to identify the risks involved in using them.

The effects of Alcea rosea L., Malva sylvestris L. and Salvia libanotica L. water extracts on the production of Anti-egg albumin antibodies, interleukin-4, gamma interferon and interleukin-12 in BALB/c mice

Polysaccharides obtained from certain plants have been reported to have immunomodulatory properties. As a consequence of these reports the aim of this study was to investigate some immunomodulatory properties of water extracts of Alcea rosea L. (ARE), Malva sylvestris L. (MSE) and Salvia libanotica L. (SLE).