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  • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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  • A61K31/431—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems containing further heterocyclic rings, e.g. ticarcillin, azlocillin, oxacillin
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  • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
  • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
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Definitions

• Chlamydiaceae comprises two genera, Chlamydia and Chlamydophila, each comprising several species; each species consists of different biovars and serovars. Each of these species leads to a set of serious pathologies in humans or animals.
• the bacteria of the family Chlamydiaceae are Gram-negative bacteria. They are called "strict intracellular” because they can only grow and multiply in a parasitophorous vacuole within an infected eukaryotic cell. During their development, bacteria of the family Chlamydiaceae come in two forms:
• the CE enters the host cell within a gallbladder. If multiple ECs infect the same cell, the vesicles cluster at the centrosome and merge with each other to form the inclusion. After 5 to 12 hours of infection, ECs differentiate into CRs that actively multiply. After 30 to 40 hours, the CRs become EC; the inclusion and the host cell are then lysed, releasing the bacterial offspring that will infect the neighboring cells. All of these parameters may vary from one serovar and one bacterial species to another, and depending on the experimental conditions (Fields, K. A. & T. hackstadt, Annu Rev Cell Dev Biol 18: 221-45, 2002).
• the Chlamydiaceae bacterium notably inhibits the apoptosis induced by external signals, inhibits the fusion of lysosomes with bacterial inclusion, defuses the vesicular traffic towards the vacuole to allow the growth of this one, and injects into the cytoplasm a key protease ( chlamydia protease / proteasome-like activity factor, CPAF) responsible for the degradation of many eukaryotic proteins, including transcription factors of major histocompatibility complex, responsible for the presentation of antigens to the immune system.
• chlamydia protease / proteasome-like activity factor, CPAF chlamydia protease / proteasome-like activity factor
• Chlamydia trachomatis which is subdivided into 2 biovars (Trachoma and Lymphogranulomatum) and 18 serovars (A to L), is particularly responsible for a wide spectrum of diseases worldwide.
• Chlamydia trachomatis are by far the leading cause of sexually transmitted infections of bacterial origin.
• Biovar LGV is responsible for another sexually transmitted disease, lymphogranuloma venereum or Durand-Nicolas-Favre disease.
• genital chlamydia are very often asymptomatic and, therefore, untreated.
• Chlamydia trachomatis can reach all levels of the genital tract and cause endocervitis in the cervix where it is a reservoir of infection. Chlamydia trachomatis can also cause urethritis and endometritis.
• Chlamydia trachomatis is the most common cause of typical or atypical mute salpingitis that often occurs during complications. Salpingitis is the main risk factor for tubal infertility and ectopic pregnancy in women. It should be noted that the fallopian tube affects the reproductive capacity of the woman several months after infection with Chlamydia trachomatis. So there is a period when fertilization and passage of the fertilized egg is possible. The egg then arrives in a contaminated uterine cavity. In this case, implantation defects, in utero growth retardation and premature labor by inflammation of the chorion were found.
• serovars A to C of Chlamydia trachomatis are responsible for an eye injury, trachoma, responsible for acquired blindness.
• Primary infection is the cause of mucopurulent conjunctivitis that causes no sequelae.
• Repeated or persistent episodes of infection result in chronic inflammation characterized by the presence of subepithelial follicles followed by papillary hypertrophy of the tarsal conjunctiva of the upper eyelid.
• the involvement of this connective tissue can progress for years resulting in an inflection of the eyelashes to the eye, causing irritation of the cornea (trichiasis) resulting in a responsible opacification of blindness.
• Vaccination is now considered the best way to control Chlamydiaceae infections. Nevertheless, the search for an anti-Chlamydiaceae vaccine is a complex task that requires finding a balance between effective protective immunity and the pathology associated with that response. On the other hand, the persistent forms of Chlamydiaceae being undetectable by the immune system, it is unlikely that a vaccine can eradicate them.
• Chlamydiaceae infections there is therefore still a need for effective treatment against Chlamydiaceae infections and, in particular, against persistent Chlamydiaceae infections.
• the present inventors have surprisingly found that persistent forms of Chlamydiaceae are ⁇ -lactam sensitive and that ⁇ -lactams can therefore be used to treat chlamydial infections.
• penicillin G far from inducing the persistence of Chlamydiaceae
• bacteria of the family of Chlamydiaceae treated with penicillin G lose all infectivity, even after the antibiotic has been removed from the medium.
• This therapeutic effect is not limited to penicillin G, but can be observed with at least one set of ⁇ -lactams. Said therapeutic effect is also not restricted to a particular bacterial species, but can be obtained with any bacterium of the family Chlamydiaceae.
• the subject of the invention is therefore a method for treating a ⁇ -lactam with an infection of a bacterium of the family Chlamydiaceae.
• the subject of the invention is also a method for treatment with a ⁇ -lactam of a pathology caused by infection with a bacterium of the family Chlamydiaceae.
• said bacterium is a bacterium of the species Chlamydia trachomatis, of the species Chlamydophila pneumoniae or of the species Chlamydophila psittaci.
• said bacterium is a bacterium of the species Chlamydia trachomatis.
• the subject of the invention is the use of a ⁇ -lactam for the manufacture of a medicament for treating infections with a bacterium of the family Chlamydiaceae.
• the subject of the invention is also the use of a ⁇ -lactam for the manufacture of a medicament for treating pathologies caused by infection with a bacterium of the family Chlamydiaceae.
• said bacterium is a bacterium of the species Chlamydia trachomatis, of the species Chlamydophila pneumoniae or of the species Chlamydophila psittaci.
• said bacterium is a bacterium of the species Chlamydia trachomatis.
• the invention also relates to a ⁇ -lactam for use in the treatment of infections with a bacterium of the family Chlamydiaceae.
• the subject of the invention is also a ⁇ -lactam for use in the treatment of pathologies caused by infection with a bacterium of the family Chlamydiaceae.
• said bacterium is a bacterium of the species Chlamydia trachomatis, of the species Chlamydophila pneumoniae or of the species Chlamydophila psittaci.
• said bacterium is a bacterium of the species Chlamydia trachomatis.
• ⁇ -lactam means any antibiotic which contains a ⁇ -lactam nucleus in its molecular structure. Included in the ⁇ -lactams for the purposes of the invention are penicillin derivatives as well as cephalosporins, monobactams, carbapenems or ⁇ -lactamase inhibitors.
• the ⁇ -lactam according to the invention may be benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), ampicillin (penicillin A), benzyl benzylpenicillin, methicillin, dicloxacillin, flucloxacillin, co -amoxiclav (amoxycillin + clavulanic acid), piperacillin, ticarcillin, azlocillin, carbenicillin, cephalexin, cephalothin, cephazolin, cefaclor, cefuroxime, cefamandole, cefotetan, cloxacillin, cephadroxil, cefexime, cefoxitin, ceftriaxone, cefotaxime, ceftazidime, cefepime, cefpirome, imipenem, imipenem in combination with cilastatin, cefixime in combination with imipenem, meropenem, mecillinam, ertapenem
• said ⁇ -lactam is not amoxicillin.
• said ⁇ -lactam is chosen from the group consisting of amoxicillin, benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), cloxacillin, cephadroxil, cefexime, imipenem, cefixime in combination with imipenem, mecillinam, clavulanic acid, tazobactam and sulbactam.
• said ⁇ -lactam is penicillin G.
• the invention therefore relates to the use of penicillin G for the manufacture of a medicament for treating infections with a bacterium of the family Chlamydiaceae or for treating pathologies caused by infection with a bacterium of the genus Chlamydiaceae.
• family of Chlamydiaceae characterized in that said treatment results in an irreversible loss of infectivity of said bacterium.
• This infectivity can be measured by any method for this purpose which is available to the skilled person. These methods have already been described in the prior art (see for example Verbeke, P. et al., PLoS Pathog 2: e45, 2006); it is not necessary to detail them further here; an example of such a method is given in the experimental examples.
• treatment with penicillin G results in degradation of the bacteria by means of a fusion with the lysosomes.
• Another more particular aspect of the invention thus consists in the use of penicillin G to manufacture a medicament for treating infections with a bacterium of the family of Chlamydiaceae or for treating pathologies caused by infection with a bacterium of the family Chlamydiaceae characterized in that said treatment causes the degradation of said bacterium.
• the degradation of said bacterium results from the fusion of the bacterium with the lysosomes.
• penicillin G is capable of inducing the degradation of bacteria of the family Chlamydiaceae.
• Chlamydiaceae family is intended to mean the taxonomic group composed of two genera, Chlamydia and Chlamydophila, as defined in Bush & Everett, Int J Syst Evol Microbiol. 51 (Pt 1): 203-20, 2001.
• Genus Chlamydia is meant within the meaning of the invention the taxonomic set comprising the species Chlamydia trachomatis, Chlamydia suis and Chlamydia muridarum, and by "genus Chamydophila The taxonomic set consisting of Chlamydophila abortus, Chlamydophila psittaci, Chlamydophila caviae, Chlamydophila pecorum, Chlamydophila felis and Chlamydophila pneumoniae (Bush & Everett, Int J Syst Evol Microbiol, 51 (Pt 1): 203-20, 2001).
• Chlamydia trachomatis and Chlamydophila pneumoniae are responsible for infections in humans, Chlamydia suis in pigs and Chlamydophila abortus in ruminants.
• the invention is not limited to a specific bacterial species, but can be applied to any of the species of the family Chlamydiaceae and, in particular, to those mentioned above.
• penicillin G is capable of inducing the degradation of Chlamydia trachomatis, regardless of its biovar or even its serovar.
• Chlamydia trachomatis is meant in the sense of the invention as well the biovar trachoma bacteria as those of the biovar lymphogranuloma venerum.
• the invention is therefore not restricted to a particular serovar, but may be used to treat infections by any of the 18 known serovars (A to L).
• the invention can be used to treat both infections with one of serovars A-C and one of serovars D to K and L1 to L3.
• infection with a bacterium of the family Chlamydiaceae or “infection with a Chlamydiaceae” means the presence in at least one cell of the body of the patient or the animal of said bacterium family Chlamydiaceae.
• the infection can be genital, ocular or pulmonary; it can also affect other sites, such as endothelium or joints.
• Said bacterium may be present in CE form or CR; it can still be present in persistent form. In a more particular aspect of the invention, the bacterium is present in persistent form in the body of the patient.
• Chlamydiaceae Infections with bacteria in the family Chlamydiaceae are not known to cause symptoms. For example, it is estimated that most cervico-vaginal infections are asymptomatic or only result in the appearance of minor symptoms (Paavonen and Eggert-Kruse, Hum Reprod Update 5 (5): 433-47, 1999) . Nevertheless, the presence of Chlamydiaceae bacteria can be detected by any means well known to those skilled in the art and it is not necessary to recall here.
• the subject of the present invention is therefore, in a particular aspect, a method for treating a ⁇ -lactam with a Chlamydiaceae infection or a pathology caused by a Chlamydiaceae infection comprising a step of detecting Chlamydiaceae. More particularly, this detection step comprises an amplification of a bacterial nucleic acid by PCR, LCR or RT-PCR.
• Chlamydia trachomatis and Chlamydophila pneumoniae are responsible for infections in humans, Chlamydia suis in pigs and Chlamydophila abortus in ruminants.
• the invention can therefore be applied to treat infections caused by Chlamydiaceae in humans as well as in animals.
• Chlamydiaceae infection pathology caused by a Chlamydiaceae infection is meant within the meaning of the invention any disease whose onset is directly or indirectly caused by infection with a bacterium of the family Chlamydiaceae.
• Chlamydiaceae in the sense of the invention are thus more particularly included in the abortions and neonatal mortalities caused by Chlamydophila abortus, as well as conjunctivitis, keratoconjunctivitis, purulent rhinitis, enteritis, bronchopneumonia. and pneumonia caused in pigs by Chlamydia suis.
• Chlamydophila pneumoniae infections cause a form of pneumonia and that Chlamydophila psittaci is responsible for psittacosis.
• infections by bacteria of the family Chlamydiaceae disseminate in the body causing many infections and atypical pathologies such as cardiovascular and circulatory dysfunctions (atheroma).
• cardiovascular and circulatory dysfunctions caused by the bacteria of the family Chlamydiaceae, there may be mentioned respectively valvary stenosis and atheroma.
• said bacteria of the family Chlamydiaceae can also cause severe arthritis.
• the pathologies caused by a Chlamydia infection are ocular pathologies or genital pathologies.
• ocular pathologies caused by Chlamydia infections include trachoma.
• the genital diseases caused by Chlamydia include lymphogranuloma venereum or Durand-Nicolas-Favre disease.
• the pathologies Genital infections in humans caused by Chlamydia include pathologies such as urethritis and orchi-epididymitis. These can lead to a decrease in male fertility.
• chlamydial infections cause in the female genital pathologies such as cervico-vaginitis, cervicitis, endocervites, urethritis, endometritis or peri-hepatitis, which can complications such as high genital infections and, in particular, salpingitis which may evolve, among other things, towards the formation of pyosalpinx and hydrosalpinx, which are responsible for the complete obstruction of the fallopian tubes.
• Salpingitis is the main risk factor for tubal infertility and ectopic pregnancy in women.
• penicillin G While doxycycline only allows partial improvement of salpingitis, treatment with penicillin G according to the invention prevents the occurrence of hydrosalpinx caused by a genital Chlamydia infection.
• Penicillin G treatment according to the invention is capable of preventing tissue damage due to Chlamydia infection. Said treatment with penicillin G according to the invention therefore shows superior efficacy compared to treatment with doxycycline, which is currently the standard treatment in humans.
• Genital diseases can be caused by infection with Chlamydia alone; however, they can also be caused by the combination of Chlamydia infection and infection by another infectious organism.
• infectious organisms capable of causing said pathologies are protozoa with Trichomonas vaginalis; fungi with Candida albicans; the proliferation of anaerobic bacteria (Bacteroides, Peptostreptococcus, Gardnerella vaginalis, Gardnerella mobiluncus) during bacterial vaginitis, for example; other bacteria such as Haemophilus ducreyi, Mycoplasma hominis, Streptococcus, Escherichia coli, Staphylococcus, Neisseria gonorrhoeae; and viral infections, for example, with Herpes simplex virus.
• the infectious organism capable of causing a genital pathology as described above is Neisseria gonorrhoeae.
• the infectious organism capable of causing a genital pathology as described above is Trichomonas vaginalis.
• the present invention therefore also relates to a method for treatment with a ⁇ -lactam of a genital pathology caused by infection with a bacterium of the family Chlamydiaceae, characterized in that said ⁇ -lactam is administered with another therapeutic agent.
• the said other therapeutic agent is chosen from the agents making it possible to treat Neisseria gonorrhoeae infections.
• said other therapeutic agent is chosen from the agents making it possible to treat Trichomonas vaginalis infections.
• Said other therapeutic agent is in particular chosen from the group consisting of ceftriaxone, cefixime, spiramycin, spectinomycin, azythromycin, ofloxacin, ciprofloxacin, metronidazole, tinidazole and mimorazole.
• the invention also relates to pharmaceutical compositions containing as active principle a ⁇ -lactam.
• Said ⁇ -lactam may or may not be associated with another therapeutic agent as defined above.
• compositions can be administered orally, rectally, parenterally or locally by topical application to the skin and mucous membranes, but the preferred route of administration is the oral route.
• compositions can be solid or liquid and be in the pharmaceutical forms commonly used in human medicine, for example, tablets, single or coated tablets, capsules, granules, suppositories, injectables, eye drops, ointments, creams, gels; they are prepared according to the usual methods.
• the active ingredient (s) can be incorporated into excipients usually employed in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non aqueous vehicles. fatty substances of animal or vegetable origin, paraffinic derivatives, glycols, various wetting, dispersing or emulsifying agents, preservatives.
• excipients usually employed in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non aqueous vehicles.
• the dose administered varies according to the condition being treated, the subject, the route of administration and the product under consideration. It may be, for example, between 50 ⁇ g and 300 mg per day orally, in adults.
• HeLa cells were infected with L2 serovar of Chlamydia trachomatis and treated with IFN ⁇ (100 ng.mL- 1 ) or penicillin G (100 U.ml- 1 ) or gentamycin (25 ⁇ g-L- 1 ). At different times after infection (hours after infection or hours post infection, hpi), the cells were fixed and labeled with an antibody against Chlamydia sp. (Green in the original figure) and with the Hoechst (blue in the original figure.) Infected HeLa cells treated with penicillin G have abnormal bodies that grow larger and are phenotypically very different from those formed during treatment with IFNy.Bodies formed with penicillin G are significantly different from persistent bodies " classics ".
• A HeLa cells were infected with Chlamydia trachomatis and treated or not with penicillin G at 3 hpi. At 21 h, staurosporine was added to the culture medium. The cells were fixed at 48 hpi, and labeled with an antibody against Chlamydia sp. (green in the original figure) and Hoechst (blue in the original figure).
• B Normal or apoptotic nuclei associated with the inclusions were counted, and percentages of apoptotic infected cells were calculated.
• Figure 4 Treatment with penicillin G results in a lack of expression of 16S rRNA.
• RNAs and cDNAs were amplified by PCR with primers for 16S (bacterial) and 18S (eukaryotic) rRNAs, to study bacterial activity and to verify the quality and quantity of the cDNAs. PCR from the RNAs verifies the absence of contamination by DNA. It also indicates the absence of contaminant in the PCR. Infected cells treated with penicillin G express 18S RNA, but not 16S, indicating that abnormal bacterial forms are not viable.
• HeLa cells were infected with Chlamydia trachomatis and either fixed and labeled (A) or immediately observed (B and C) at 24 hpi.
• A Hoechst marking (blue in the original figure), anti-Chlamydia sp. (green in the original figure) and anti-cathepsin D (red in the original figure). Cathepsin D is remarkably present in abnormal bodies. Scale: 10 ⁇ .
• B The cells were incubated with the lysotracker for 30 min. The lysotracker is located mainly in abnormal inclusions with a complex membrane network. The details show highly shiny spots in abnormal inclusions, suggesting the entry of a lysosome into an abnormal inclusion. Arrow head: inclusion membrane, arrow: compartment loaded with lysotracker. DIC: differential interference contrast. Scale: 10 ⁇ .
• a monocyte / macrophage tumor cell line, THP-1, an endometrial tumor cell line, RL95-2, and a cervical tumor cell line, HeLa, infected with the serovar L2 of the biovar LGV from Chlamydia trachomatis, or by the serovar D of the Trachoma biovar, or by Chlamydia muridarum, and treated with penicillin G (3 hpi) have abnormal inclusions.
• Hoechst in green in the original figure: anti-Chlamydia sp. Image taken at 24 hpi (RL95-2 / Chlamydia L2; H / Chlamydia-D; HeLa / C. Muridarum) or at 48 hpi (THP-IChlamydia-L2; ⁇ -1 / Chlamydia-D). Scale: 10 ⁇ .
• HeLa cells were infected in the absence (white bar) or in the presence of penicillin G (100 U.ml "1 ) added at 2 hpi (light gray bar) or 29 hpi (dark gray bar) Staurosporine was added at 31 hpi The cells were fixed and observed as described below The apoptotic or normal nuclei associated with the inclusions were counted, and the percentages of cells infected with apoptosis were determined. Penicillin G induces a loss of control of Chlamydia trachomatis on apoptosis of the host cell.
• penicillin G was added at 1, 10 or 100 U.ml- 1 in penicillin G (-) samples and reversion (+
• penicillin G was removed from the culture medium in the (+) reversion group, at 48 hpi or 100 hpi supernatants and cells were harvested and stored at -80 ° C.
• HeLa grown on slides were incubated for 1 h with cell extracts (left) or supernatants (right), treated with cycloheximide and fixed 24 h later. The nuclei and inclusions were labeled and counted to determine the number of IFU.ml "1 . Under control conditions (bacterial offspring from untreated cells), significant infectivity was detected, whereas no reinfection was achieved with bacteria collected from cells treated permanently or transiently with penicillin G.
• Cathepsin D is localized in the abnormal forms of Chlamydia trachomatis independently of the biovar and the type of host cell.
• Figure 10 Effect of a treatment with penicillin G on the development of hydrosalpinx following the infection of C57B1 / 6 mice by Chlamydia muridarum,
• mice were infected by the vaginal route with 10 7 IFUs of Chlamydia muridarum. After ten days, one group of mice received no antibiotic (untreated), another was treated with doxycycline 5mg / ml orally and the last group received penicillin G at 5mg / ml per os. Antibiotic treatment was discontinued after 20 days and the mice were kept isolated for an additional 60 days. The mice have then sacrificed, the appearance of the genital tract analyzed and the presence of hydrosalpinx (arrows) sought.
• Recombinant human IFN- ⁇ , cycloheximide, 3-methyl-adenine (3-MA), staurosporine and penicillin G were obtained from Sigma-Aldrich (St. Louis, MO, USA).
• the lysotracker also comes from Invitrogen (Carlsbad, CA, USA).
• a mixture of two mouse monoclonal antibodies against the Chlamydia genus and FITC conjugates were obtained from Argen Biosoft (Varhilles, France).
• Polyclonal rabbit (IgG) antibodies against cathepsin D and Texas Red conjugated goat anti-rabbit antibodies are from Santa Cruz (Santa Cruz Biotechnology, CA, USA). Control rabbit IgG were purified from rabbit serum using protein A-sepharose.
• HeLa, RL-95.2, THP-1 All cell types (HeLa, RL-95.2, THP-1) were grown according to the American Type Culture Collection guidelines (ATCC, Manassas, Va., USA), in 75 cm 2 culture flasks for maintenance. and in 12- or 24-well plates with lids or in Lab-Tek TM culture chambers for experiments. The differentiation of THP-1 cells into macrophages was obtained using PMA at 0.25 ⁇ in the culture medium.
• the cultures were either fixed in 4% buffered PFA at neutral pH for 30 min to be prepared for confocal microscopy, or harvested to determine the infectivity of the bacterial offspring, according to the method previously described. (Verbeke, P. et al., PLoS Pathog 2: e45, 2006).
• HeLa cells grown in slide monolayers were infected with L2 serovar at an IFU of 1, as previously described (Verbeke, P. et al., PLoS Pathog 2: e45, 2006). Three hours after infection, the medium was removed and replaced with culture medium containing recombinant human IFN-gamma at 500 IU.ml- 1 (final concentration) The infected cells were incubated for an additional 100 hours in the presence of IFN- ⁇ , before being prepared for confocal microscopy as described below 2.3 Reactivation test
• HeLa cells grown in slide monolayers were infected with Chlamydia trachomatis L2 serovar and treated with penicillin G as described above. Twenty hours after infection, the culture was washed and the medium replaced with medium not containing penicillin G. Between 48 and 100 hpi, the cells and the culture medium were harvested at different intervals and a titration test was conducted according to the method of the prior art (Scidmore, MA Curr Microbiol Chapter Protoc ll: Unit 11A 11, 2005).
• L2-infected HeLa cells treated with IFN-gamma G with penicillin G (500 IU.mr 1 ) of 48 hpi (24 h after treatment with IFN- ⁇ ) up to 72 hpi (48h after IFN- ⁇ treatment).
• the slides were then fixed and observed using an epifluorescence microscope (Leica DM, Leica, Mannheim, DE) equipped with two sets of detection filters (Hoechst 360 / 40nmBP-425nmLP, FITC 480 / 40nmBP-527 / 30nmBP) and connected to a CCD camera.
• HeLa cells cultured in slide monolayers were infected with serovar L2 and treated with penicillin G at 3 hpi or 29 hpi. At 25 hpi or 32 hpi, respectively, the cells were incubated with 1 ⁇ l staurosporine, fixed at 40 hpi or 48 hpi and prepared for epifluorescence microscopy. Percentages of infected cells containing apoptotic nuclei were calculated. 2.5. Confocal microscopy
• HeLa cells cultured in slide monolayers were infected with serovar L2. At 3 hpi, the cells were or were not treated with penicillin G. Then, the cells were fixed at 24 hpi, permeabilized and incubated (2 h, room temperature) with an anti-cathepsin D antibody (1/50). PBS-BSA 1%. After several washings, a Texas Red conjugated goat anti-rabbit IgG (1/200) was added to the slides (1h, room temperature). Then the cells were countermarked for 1h with FITC-conjugated anti-chlamydial antibody (1/800) and 5 min with Hoechst (1/2000).
• the slides were then mounted and observed using a confocal microscope (Leica, Tandem TCS Sp5 AOBS, Leica, Mannheim, DE) equipped with two laser diodes (1 and 25nW) emitting at 405nm and 561nm respectively. an argon laser (100nW) emitting at 488 nm. Emitted signals were collected at 411-481 nm for Hoechst, 493-555 nm for FITC and 591-703 nm for Texas Red. Each experiment was conducted, acquired and analyzed in a similar fashion, and was repeated three times.
• RNAs were isolated using the RNeasy Mini Plus kit (Qiagen, Hilden, DE). The samples were treated with DNase I for 1 hour at 37 ° C. The enzyme was then denatured at 70 ° C for 10 minutes. The RNAs were retranscribed in cDNA using random hexameric primers and reverse affinity script MT (Agilent, Santa Clara, CA, USA) and following the manufacturer's instructions for RT-PCR. The cDNAs and the RNAs were amplified by PCR with Taq Polymerase (Invitrogen, Carlsbad, CA, USA) to verify the absence of contamination by DNA.
• Taq Polymerase Invitrogen, Carlsbad, CA, USA
• the primers used to amplify the 16S rRNA are:
• the primers used to amplify eukaryotic 18S rRNA are:
• mice C57B1 / 6 mice were infected with Chlamydia muridarum, a Chlamydia species that infects mice and guinea pigs and is genetically close to Chlamydia trachomatis.
• the mice were cycled by injection of progesterone and then infected by the vaginal route after 3 days (10 7 IFU).
• Ten days after infection the mice were divided into three groups, a negative control group receiving no antibiotics, a positive control group receiving doxycycline (5 mg / ml) and a test group receiving penicillin G (5 mg / ml). ml).
• the duration of treatment was 20 days; antibiotics, doxycycline and penicillin G were added to the drinking water of the mice.
• the mice were kept isolated for 60 days to allow persistent bacteria to resume their cycle and sequelae to develop. The mice were then sacrificed, the appearance of the genital tract analyzed and the presence of hydrosalpinx sought. Different organs were also collected.
• 16S ribosomal AR expression It is known that both CR and the persistent form of Chlamydia express 16S ribosomal RNA (rRNA), which is essential for the bacterium.
• rRNA 16S ribosomal RNA
• the expression of this 16S rRNA was studied in the abnormal bacterial forms induced by penicillin G.
• the RNAs were extracted from infected cells treated with penicillin G, gentamicin or IFN- ⁇ , and 16S rRNA expression was studied by RT-PCR.
• a specific sequence of 16S rRNA could thus be amplified from infected cells treated with gentamicin (FIG. 4) or with IFN- ⁇ .
• infected cells treated with penicillin G do not produce bacterial RNA.
• all infected cells all expressed the same levels of eukaryotic 18S rRNA, indicating that RNA extraction and RT-PCR were effective.
• no contamination by DNA was observed by PCR on the extracted RNAs.
• these results show that CR and the persistent form of Chlamydia produce 16S rRNA.
• the bacteria treated with penicillin G have lost the ability to express this essential RNA.
• penicillin G causes Chlamydia degradation. Intracellular particles confined in vesicles are sent to the lysosome to be degraded. The possibility that abnormal inclusions fuse with the lysosome has been studied.
• Cathepsins are proteases that play an important role in the breakdown of proteins by the lysosome.
• cathepsin D is a lysosomal aspartic protease.
• the localization of cathepsin D has been studied in infected cells treated with IFN ⁇ and penicillin G or not.
• the majority of abnormal bacterial forms contained cathepsin D ( Figure 5A).
• this marker was excluded from normal and persistent inclusions.
• no labeling was found.
• the lysotracker is a probe used to detect acidic compartments such as lysosomes.
• the retention mechanism of the lysotracker involves its protonation which leads to its localization in the membranes of organelles (Haugland, RP The Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 10th Edition, 2005. Invitrogen Corp., Spence MTZ, 580-588 pp). Labeling of cells infected with this probe revealed a complex tubular network in abnormal inclusions and only in abnormal inclusions. In addition, very strong points have been observed in the majority of these abnormal inclusions (Figure 5B, box). These points could be lysosomes penetrating into an abnormal inclusion.
• penicillin G The effects of penicillin G have been tested on persistent infections.
• the cells were infected and then treated with IFN- ⁇ at 3 hpi. Once the appearance of persistent inclusions was observed under the microscope, penicillin G was added to infected cells cultured in the presence of IFN- ⁇ . A rapid restart of inclusion growth was then observed, as well as an abnormal expansion of bacterial particles in said inclusions. Twenty-four hours after the addition of penicillin G, the majority of abnormal bacterial forms contained cathepsin D. Finally, the cells lysed 70 h after the start of treatment with penicillin G, lysis accompanied by the release of non-invasive bacteria. infectious. This result shows that treatment with penicillin G leads to the degradation of persistent forms of Chlamydia.
• mice infected with penicillin G leads to absence of hydrosalpinx
• penicillin G has been tested in a mouse model of genital infection. In this model, the formation of hydrosalpinx in the uterine horns is observed. These inflammatory cysts very commonly observed in mammals in case of pelvic infections by Chlamydia trachomatis result from the joining of the walls and the collection of liquid.
• PenicillinV potassium knows 46616 0
• Cloxacillin sodium knows hydrate 46140 0
• Tazobactam sodium knows T2820 0

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