Pierrette Gourde

Infection of Primary Human Monocytes by Epstein-Barr Virus

ABSTRACT Previous studies have reported that infection of monocytes by viruses such as cytomegalovirus and human immunodeficiency virus weakens host natural immunity. In the present study, we demonstrated the capability of Epstein-Barr virus (EBV) to infect and replicate in freshly isolated human monocytes. Using electron microscopy analysis, we observed the presence of EBV virions in the cytoplasm and nuclei of approximately 20% of monocytes. This was confirmed by Southern blot analysis of EBV genomic DNA sequences in isolated nuclei from monocytes. Infection of monocytes by EBV leads to the activation of the replicative cycle. This was supported by the detection of immediate-early lytic mRNA BZLF-1 transcripts, and by the presence of two early lytic transcripts (BALF-2, which appears to function in DNA replication, and BHRF-1, also associated with the replicative cycle). The late lytic BcLF-1 transcripts, which code for the major nucleocapsid protein, were also detected, as well as EBNA-1 transcripts. However, attempts to detect EBNA-2 transcripts have yielded negative results. Viral replication was also confirmed by the release of newly synthesized infectious viral particles in supernatants of EBV-infected monocytes. EBV-infected monocytes were found to have significantly reduced phagocytic activity, as evaluated by the quantification of ingested carboxylated fluoresceinated latex beads. Taken together, our results suggest that EBV infection of monocytes and alteration of their biological functions might represent a new mechanism to disrupt the immune response and promote viral propagation during the early stages of infection.

Molecular basis of the non-beta-lactamase-mediated resistance to beta-lactam antibiotics in strains of Haemophilus influenzae isolated in Canada.

A study recently conducted across Canada showed that 64 of 2,503 clinical isolates of Haemophilus influenzae were resistant to beta-lactams without production of a beta-lactamase (L. D. Tremblay, J. LEcuyer, P. Provencher, M. G. Bergeron, and Canadian Study Group, Can. Med. Assoc. J. 143:895-900, 1990). The beta-lactamase-negative strains formed three distinct groups, with ampicillin MICs of 0.5 to 1, 2 to 4, and greater than or equal to 8 micrograms/ml for groups I, II, and III, respectively. We have investigated the mechanisms of resistance for eight strains originating from different infections and geographic areas. These strains were representative of groups I to III. Five strains were nontypeable, two were type B, and one was non-B. Chromosomal DNA extracted from each strain was used to transform the laboratory strain Rd. Transformants were selected on beta-lactam-containing plates and showed the same level of resistance to ampicillin as the donor strains. Differences in outer membrane proteins, porins, and lipopolysaccharide profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) did not change with resistance. Functional analyses of purified porins in artificial lipid bilayer experiments did not explain resistance. Peptidoglycan synthesis was measured by incorporation of [14C]alanine into trichloroacetic acid-insoluble cell wall material in the presence of chloramphenicol. The growth rate and the rate of peptidoglycan synthesis observed for the transformants of the isogenic set did not correlate with resistance. Whole-cell labeling with 125I-penicillin revealed modifications in penicillin-binding proteins (PBPs) among the transformants. In particular, PBPs 3A and 3B (65 and 63 kDa, respectively) showed a decrease in affinity for beta-lactams in all transformants (groups I, II, and III) and correlated with an increased MIC except in the transformant of group III, which showed higher levels of resistance. Partial purification and proteolytic digestion of 125I-penicillin-labeled PBP 3B led to two types of CnBr peptide profiles on SDS-PAGE, the profiles of the transformed strains from groups I and II being different from those of the control group and group III. Finally, electron microscopy revealed a distinct cell filamentation for the group III transformants. These data clearly indicate that changes in PBPs are a common mechanism that results in a significant level of non-beta-lactamase-mediated beta-lactam resistance in H. influenzae despite serotype, origin of isolation, or geographic distribution. Images

Subcellular distribution of gentamicin in proximal tubular cells, determined by immunogold labeling.

The subcellular distribution of gentamicin in rat renal proximal tubular cells was evaluated by immunogold labeling. The distribution of the drug was monitored from 10 min to 10 days following single (40 mg/kg of body weight) and multiple (5 and 20 mg/kg/12 h) injections of gentamicin. Animals were killed on day 11, and cubes of renal cortex tissue were fixed overnight in cold phosphate-buffered glutaraldehyde (0.5%), dehydrated in ethanol, and embedded in Araldite 502 epoxy resin. Ultrathin sections were made and incubated with sheep antigentamicin and then with protein A-gold (15 nm) complex. At 10 min after a single injection, the labeling was found over the brush border membrane and over the membranes of endocytic apical vesicles of proximal tubular cells. After 1 h, a similar distribution was observed and the labeling was also seen over small lysosomes located close to the brush border membrane. At 24 h, gold particles were found over large lysosomes of proximal tubular cells. Following 10 days of treatment, lysosomes of proximal tubular cells were densely labeled with gold particles. The labeling was distributed uniformly over the lysosomes, although a lower density of labeling was observed over the myeloid bodies inside the lysosomes. Necrotic proximal tubular cells showed labeling over intact lysosomes and also in the cytoplasms of the cells, in the mitochondria, and in the nucleoli. The various control experiments demonstrated the high specificity of these results. The present immunocytochemical study better documents the subcellular disposition of gentamicin in proximal tubular cells, as previously evaluated by subcellular fractionation and autoradiography. This technique will be useful for better understanding the relationship between drug disposition and drug-induced toxicity. Images

Effects of daptomycin and vancomycin on tobramycin nephrotoxicity in rats.

Daptomycin is a new biosynthetic antibiotic which belongs to a new class of drugs known as lipopeptides. The objective of this study was to evaluate the effects of daptomycin and vancomycin on tobramycin-induced nephrotoxicity. Female Sprague-Dawley rats were treated during 4 and 10 days with either saline (NaCl, 0.9%) or tobramycin at doses of 4 and 40 mg/kg per day (given every 12 h [q12h] intraperitoneally). Each treatment was combined with saline, daptomycin at a dose of 20 mg/kg per day (given q12h subcutaneously), and ancomycin at a dose of 50 mg/kg per day (given q12h subcutaneously). Daptomycin and vancomycin had no effect on the intracortical accumulation of tobramycin. Daptomycin did not accumulate in renal tissue even after 10 days of treatment. Tobramycin given at a dose of 40 mg/kg per day during 10 days induced a significant inhibition of sphingomyelinase activity in the renal cortex (P less than 0.01) and increased cellular regeneration (P less than 0.01), as measured by the incorporation of [3H]thymidine into DNA of the renal cortex. These changes were minimal when daptomycin was combined with tobramycin. Histologically, signs of tobramycin toxicity were also less severe in the presence of daptomycin. The intracortical accumulation of vancomycin was not modified by tobramycin. The sphingomyelinase activity was significantly more inhibited (P less than 0.01) when vancomycin was associated with tobramycin (4 and 40 mg/kg) without affecting the rate of [3H]thymidine incorporation into DNA. Histologically, signs of tobramycin toxicity were not affected by vancomuycin, but the cellular vacuolizations which were also observed in vancomycin-treated animals were still present in the proximal tubular cells of animals that were treated with the combination vancomycin-tobramycin. This study strongly suggests that daptomycin protects animals from tobramycin-induced nephrotoxicity but that vancomycin may enhance the effect of tobramycin. We conclude that daptomycin is safe and protects kidney cells from tobramycin-induced nephrotoxicity. Images

Thermoreversible Gel Formulations Containing Sodium Lauryl Sulfate or n-Lauroylsarcosine as Potential Topical Microbicides against Sexually Transmitted Diseases

ABSTRACT The microbicidal efficacies of two anionic surfactants, sodium lauryl sulfate (SLS) and n-lauroylsarcosine (LS), were evaluated in cultured cells and in a murine model of herpes simplex type 2 (HSV-2) intravaginal infection. In vitro studies showed that SLS and LS were potent inhibitors of the infectivity of HSV-2 strain 333. The concentrations of SLS which inhibit viral infectivity by 50% (50% inhibitory dose) and 90% (90% inhibitory dose) were 32.67 and 46.53 μM, respectively, whereas the corresponding values for LS were 141.76 and 225.30 μM. In addition, intravaginal pretreatment of mice with thermoreversible gel formulations containing 2.5% SLS or 2.5% LS prior to the inoculation of HSV-2 strain 333 completely prevented the development of genital herpetic lesions and the lethality associated with infection. Of prime interest, no infectious virus could be detected in mouse vaginal mucosa. Both formulations still provided significant protection when viral challenge was delayed until 1 h after pretreatment. Finally, intravaginal application of gel formulations containing 2.5% SLS or 2.5% LS once daily for 14 days to rabbits did not induce significant irritations to the genital mucosa, as demonstrated from macroscopic and histopathologic examinations. These results suggest that thermoreversible gel formulations containing SLS or LS could represent potent and safe topical microbicides for the prevention of HSV-2 and possibly other sexually transmitted pathogens, including human immunodeficiency virus.

Targeting lymph nodes with liposomes bearing anti-HLA-DR Fab' fragments.

The ability of liposomes bearing anti-HLA-DR Fab fragments to target cells expressing the human HLA-DR determinant of the major histocompatibility complex class II (MHC-II) has been evaluated and compared to that of conventional liposomes. Anti-HLA-DR immunoliposomes did not bind to HLA-DR-negative cells. In contrast, a high level of binding was observed following incubation of immunoliposomes with cells bearing important levels of human HLA-DR. The accumulation of conventional and murine anti-HLA-DR immunoliposomes in different tissues has been investigated following a single subcutaneous injection given in the upper back of C3H mice. Anti-HLA-DR immunoliposomes resulted in a much better accumulation in the cervical and brachial lymph nodes when compared to conventional liposomes. The accumulation in the liver was similar for both liposomal preparations, whereas an approximately twofold decrease in accumulation was observed for immunoliposomes in the spleen. Given that HLA-DR surface marker is expressed on monocyte/macrophages and activated CD4+ T lymphocytes, the primary cellular reservoirs of the human immunodeficiency virus (HIV), the use of liposomes bearing surface-attached anti-HLA-DR could constitute a convenient strategy to more efficiently treat this debilitating retroviral disease. Moreover, the reported incorporation of high amounts of host-encoded HLA-DR proteins by HIV particles renders the use of liposomes bearing anti-HLA-DR antibodies even more attractive.

Sterically stabilized liposomes bearing anti-HLA-DR antibodies for targeting the primary cellular reservoirs of HIV-1

The ability of liposomes bearing anti-HLA-DR Fab fragments at the end termini of polyethyleneglycol chains (sterically stabilized immunoliposomes) to target HLA-DR expressing cells and increase the accumulation of liposomes into lymphoid organs has been evaluated and compared to that of conventional liposomes, sterically stabilized liposomes and conventional immunoliposomes after a single subcutaneous injection to mice. The accumulation of sterically stabilized liposomes in lymph nodes was higher than that of conventional liposomes. Sterically stabilized immunoliposomes accumulated much better than conventional immunoliposomes in all tissues indicating that the presence of PEG has an important effect on the uptake of immunoliposomes by the lymphatic system. Fluorescence microscopy studies showed that sterically stabilized liposomes are mainly localized in macrophage-rich areas such as the subcapsular region of lymph nodes and in the red pulp and marginal zone of the spleen. In contrast, sterically stabilized immunoliposomes mostly accumulated in the cortex in which follicles are located and in the white pulp of the spleen. As the human HLA-DR determinant of the major histocompatibility complex class II is expressed on activated CD4+ T lymphocytes and antigen presenting cells such as monocyte/macrophages and dendritic cells, known as the cellular reservoirs of HIV-1, liposomes bearing anti-HLA-DR antibodies constitute an attractive approach to concentrate drugs in HIV-1 reservoirs and improve their therapeutic effect.

Protective effect of a thermoreversible gel against the toxicity of nonoxynol-9

BACKGROUND AND OBJECTIVESnOne major problem associated with the use of nonoxynol-9 is that it can induce local inflammation and ulceration of the vaginal and cervical mucosa that might favor the entry of pathogens. With the aim of developing a gel formulation that could be effective in preventing sexually transmitted infections, the authors have evaluated the capacity of a polyoxypropylene/polyoxyethylene polymer to reduce or eliminate the toxicity of nonoxynol-9.nnnSTUDY DESIGNnThe cytotoxicity of nonoxynol-9 alone or incorporated into the gel was investigated in human cervical and colon epithelial cells and after daily intravaginal application for 2 weeks in rabbits.nnnRESULTSnIn vitro experiments showed that nonoxynol-9 was highly toxic to human cervical and colon epithelial cells in a dose-dependent manner. However, the incorporation of the spermicide into the gel markedly reduced its toxicity under the same experimental conditions. In vivo studies showed that in animals treated with nonoxynol-9, the spermicide was very toxic to the vaginal and cervical mucosa as evidenced by the presence of bleeding, irritation, epithelial disruption, necrosis, the accumulation of leukocytes in the submucosa, and the loss of integrity of the epithelial cells. Of prime importance, the incorporation of nonoxynol-9 into the gel markedly reduced the toxicity of this potent spermicide/microbicide.nnnCONCLUSIONnThe gel formulation could be used as an interesting approach to eliminate the toxicity of potent spermicides/microbicides such as nonoxynol-9.

Subcellular localization of tobramycin and vancomycin given alone and in combination in proximal tubular cells, determined by immunogold labeling.

The subcellular localization of tobramycin and vancomycin in the renal cortices of rats was determined with ultrathin sections by immunogold labeling. Four groups of four rats each were treated for 10 days with saline (NaCl, 0.9%), tobramycin at dosages of 20 mg/kg of body weight per 12 h intraperitoneally, vancomycin at dosages of 25 mg/kg/12 h subcutaneously, or the combination tobramycin-vancomycin. On day 11, the animals were killed, and cubes of renal cortex were fixed overnight in phosphate-buffered glutaraldehyde (0.5%), dehydrated in ethanol, and embedded in Araldite 502 resin. Ultrathin sections were made and incubated with sheep antitobramycin antibody followed by protein A-gold (15-nm diameter) complex or rabbit antivancomycin antibody followed by gold (30-nm diameter)-labeled goat anti-rabbit antibody. For the double labeling, incubations were made on opposite sides of the grid. Tobramycin was detected over the lysosomes of proximal tubular cells, but the labeling was concentrated into small areas in the matrix of the lysosomes. Vancomycin was seen over the lysosomes of proximal tubular cells and was distributed uniformly throughout the matrix of the lysosomes. In rats treated with tobramycin-vancomycin, both drugs were still detected in lysosomes of proximal tubular cells. It is concluded that tobramycin and vancomycin accumulate in lysosomes of proximal tubular cells throughout 10 days of treatment and that vancomycin has no effect on the subcellular distribution of tobramycin. Images

Thermoreversible Gel Formulation Containing Sodium Lauryl Sulfate as a Potential Contraceptive Device

Abstract The contraceptive properties of a gel formulation containing sodium lauryl sulfate were investigated in both in vitro and in vivo models. Results showed that sodium lauryl sulfate inhibited, in a concentration-dependent manner, the activity of sheep testicular hyaluronidase. Sodium lauryl sulfate also completely inhibited human sperm motility as evaluated by the 30-sec Sander-Cramer test. The acid-buffering capacity of gel formulations containing sodium lauryl sulfate increased with the molarity of the citrate buffers used for their preparations. Furthermore, experiments in which semen was mixed with undiluted gel formulations in different proportions confirmed their physiologically relevant buffering capacity. Intravaginal application of the gel formulation containing sodium lauryl sulfate to rabbits before their artificial insemination with freshly ejaculated semen completely prevented egg fertilization. The gel formulation containing sodium lauryl sulfate was fully compatible with nonlubricated latex condoms. Taken together, these results suggest that the gel formulation containing sodium lauryl sulfate could represent a potential candidate for use as a topical vaginal spermicidal formulation to provide fertility control in women.