Efficacy of PD-1 blockade in cervical cancer is related to a CD8+FoxP3+CD25+ T-cell subset with operational effector functions despite high immune checkpoint levels.

BACKGROUND: Cervical cancer (CxCa) is mainly a locally invading disease that metastasizes to loco-regional lymph node basins before involving distant organs in more advanced stages. Local immune potentiation of tumor-draining lymph nodes (TDLN) may thus protect against tumor progression. METHODS: To identify therapeutic targets for local immune modulation, multi-parameter flow cytometric T-cell profiling of primary cervical tumors (PT) and TDLN (n = 37) was performed. The in-vitro effect of PD-1 blockade on T-cell reactivity to HPV16 E6 oncoproteins was determined in cultures of TDLN and PT single cell suspensions (n = 19). Also, intracellular cytokine staining (ICS) upon anti-CD3 stimulation was performed in metastatic TDLN (LN+) and PT (n = 7), as well as multiplexed immunofluorescence histochemistry staining (n = 8). RESULTS: Our data revealed elevated rates of activated regulatory T cells (aTregs) and of central or effector memory CD8+ T cells in metastatic TDLN (LN+) as compared to tumor-free TDLN (LN-), and equally high or even higher rates of these subsets in PT. Both memory subsets co-expressed multiple immune checkpoints. PD-1 blockade significantly enhanced detectable E6-specific T-cell responses in 4/5 HPV16+ LN+ and in 1/5 HPV16+ PT. Whereas aTreg rates were higher in anti-PD-1 non-responders, in responders elevated levels of CD8+FoxP3+CD25+ T cells were observed, which correlated with the efficacy of PD-1 blockade (P = 0.018). This subset was characterized by an early effector memory phenotype with particularly high levels of co-expressed PD-1, CTLA-4, TIM-3 and LAG-3 checkpoints, but, rather than exhausted, was shown upon polyclonal activation to produce higher levels of Granzyme-B and effector cytokines as compared to its CD8+FoxP3- counterparts. CONCLUSION: These observations support local PD-(L)1 blockade to interrupt loco-regional immune suppression in CxCa and control metastatic spread to TDLN. Furthermore, our data identify CD8+FoxP3+CD25+ T cells as therapeutic targets, which may also serve as predictive biomarker for PD-(L)1 checkpoint blockade.

Evaluation of the use of Home Emergency in a Sub-Sahara African hospital: Example of the Hospital Principal in Dakar.

Overloaded emergency departments are common around the world. This prospective, descriptive, and analytical study evaluates the reasons for emergency room use and describes the severity of the patients' conditions and how they reach the emergency department. It took place at the emergency department of the principal hospital of Dakar (the Armed Forces Teaching Hospital) over a one-week period (from August 23 to August 30, 2016) and included a sample of 219 patients of both sexes aged 15 years and older. Data were collected about social and demographic characteristics, reasons for choosing the emergency department rather than other care, time of day, day of the week, accessibility, means of transport, diagnosis, and severity. More than half of patients (55 %) were men, with a mean age of 42 +/- 18. Only 10 % of patients lived within 5 km of the hospital. Most (84.5 %) came from the Dakar region and lived between 5 and 35 km from the hospital. Most patients reached the hospital by their own means (83 %). Only 2 % were transported by the public emergency ambulance service. The reasons for choosing the emergency unit were the opportunity for additional tests in 41.1 % of cases ; the desire to be hospitalized in 26.9 % ; fear of death in 26.5 %, no waiting list in 14.2 %, and lack of money in 11 % of cases. Patients in truly serious condition accounted for only 5 % of cases. Most (88 %) returned home after stabilization, 12 % were hospitalized, and 0.5 % died. Good practices, good organization, and improved complementarity between public, private, and emergency services are needed to reduce the use of the latter.

Syndrome occlusif au décours d'une ingestion chronique de Kaolin ("Kew"). A propos d'une observation

Le pica est un trouble du comportement alimentaire fréquemment rencontré chez les femmes et les enfants. Il se caractérise par l'ingestion d'objets ou de substances non-comestibles comme la craie, le charbon, le savon, le métal, le sable etc... Nous rapportons une observation d'une patiente de 20 ans avec notion d'autisme depuis l'enfance et d'addiction à la géophagie (Kaolin appelé "kéw" au Sénégal) depuis plusieurs années qui a eu à consulter pour des douleurs abdominales associées à des ballonnements abdominaux, une hématémèse et un arrêt complet des matières et des gaz. Son examen clinique avait objectivé un abdomen légèrement sensible sans défense ni cris de l'ombilic et le toucher rectal avait objectivé un fécalome de consistance dure. Le bilan biologique était normal en dehors d'une légère hypokaliémie, le test de Wide était négatif. La radiographie de l'abdomen sans préparation avait objectivé des micro calcifications en amas au niveau du rectum confortée par la tomodensitométrie abdomino-pelvienne qui avait en plus infirmé l'existence d'une perforation d'organe et de souffrance viscérale. Un traitement évacuateur mécanique consistant en la mise en place d'une sonde d'intubation orotrachéale charnière n°6 au niveau du rectum avec ballonnet gonflé et irrigation par 500 ml de sérum savonneux stérile suivi d'une évacuation au doigt avait permis d'évacuer la quasi-totalité des corps étrangers endorectaux confirmée par la radiographie de contrôle. Devant la constatation d'une ré-ingestion au bout d'une semaine avec une radiographie de l'abdomen sans préparation qui avait montré des images quasi-superposable, la patiente a été adressée en consultation psychiatrique pour un suivi

Ordinary and opportunistic enteropathogens associated with diarrhea in Senegalese adults in relation to human immunodeficiency virus serostatus.

OBJECTIVES: A survey was conducted in Dakar, Senegal, to identify major types and prevalences of bacteria, parasites, fungi, and Rotaviruses associated with diarrhea in relation to human immunodeficiency virus (HIV) serostatus with the goal to provide guidance to physicians for case management. METHODS: Etiologic agents were identified in a case control study: cases were HIV-infected patients with diarrhea (HIV+ D+) and HIV seronegative patients with diarrhea (HIV D+); controls were HIV-infected patients without diarrhea (HIV+ D ) and seronegative controls without diarrhea (HID D ). Ordinary enteric pathogens were identified by conventional methods. Different Escherichia coli pathotypes were characterized by polymerase chain reaction (PCR), identification of HEp-2 cell adherence pattern, Sereny test, GM1-ELISA, and the suckling mouse assay. Opportunistic parasites, such as Cryptosporidium and Microsporidium, were identified by the Kinyoun method and trichromic stain of Weber, respectively. Rotaviruses were identified with a commercial latex agglutination kit. Antimicrobial susceptibility testing was carried out by the disk diffusion method. RESULTS: Among the 594 patients examined, 158 were HIV+ D+, 121 were HIV2 D+, 160 were HIV+ D , and 155 were HIV D . The main etiologies of diarrhea were different according to HIV serostatus of patients. In immunocompetent adults the main causes of diarrhea were Shigella sp (12.4%), Entamoeba histolytica(10.7%), Salmonella enterica (6.6%), and Giardia (4.9%). In the immunocompromised host the more frequent pathogens were enteroaggregative E. coli (19.6%), Microsporidium (9.4%), Cryptosporidium sp (8.2%), Rotavirus (8.2%), Shigella sp (7.6%), Candida albicans (7.6%), E. histolytica (5.1%), S. enterica (4.4%), and Isospora belli (4.4%). Also, Blastocystis hominis has to be considered as an opportunistic parasite, because it was identified only in HIV-infected patients, with higher prevalence in adults with diarrhea (2.5% in HIV+ D+ patients; 0.6% in HIV+ D patients). High level of asymptomatic carriage of Ascaris lumbricoides and Trichuris trichiura and some cases of multiple infections were observed. Fungi, Cryptosporidium sp and Microsporidium sp, were often identified in patients with low CD4 counts (range, 79 250 cells/mL). Independently from HIV-serostatus, CD4 count was lower in diarrheic persons, suggesting that diarrhea is a debilitating illness and that effective management of diarrhea can prevent immunosuppression. Isolated enteropathogenic strains displayed high resistance to most antibiotics used in Senegal for treating diarrhea (ampicillin, tetracycline, cotrimoxazole); they were susceptible to amikacin, gentamicin, and norfloxacin. CONCLUSION: These epidemiologic data suggest that guidelines for the management of diarrhea during HIV infection in Dakar should be updated.

[Infective etiology of diarrhea in adults with HIV infection in Dakar: a case-control study on 594 patients]. / Etiologies infectieuses des diarrhées de l'adulte au cours de l'infection à VIH à Dakar: étude cas/témoins sur 594 malades.

A survey was conducted in Dakar, Senegal from May 1997 to May 1999, to identify major types and prevalences of bacteria, parasites, fungi and Rotaviruses associated with diarrhea in relation to human immunodeficiency virus serostatus with the aim to provide guidance to physicians for case management. Etiologic agents were identified in a case-control study: cases were HIV-infected patients with diarrhea (HIV+ D+) and HIV seronegative patients with diarrhea (HIV- D+); controls were HIV-infected patients without diarrhea (HIV+ D-) and seronegative controls without diarrhea (HIV- D-). Strict enteric pathogens were identified by conventional methods. Different E. coli pathotypes were characterized by PCR. Opportunistic parasites such as Cryptosporidium and Microsporidium were identified by the Kinyoun method and trichromic stain of Weber respectively. Rotaviruses were identified with a commercial latex agglutination kit. Antimicrobial susceptibility testing was carried out by the disk diffusion method. A total of 594 patients were examined (158 HIV+D+, 121 HIV- D+, 160 HIV+ D- and 155 HIV- D-). In immunocompetent adults the main causes of diarrhea were: Shigella sp. (12.4%), Entamoeba histolytica (10.7%), Salmonella enterica (6,6%) and Giardia lamblia (4.9%). In the immunocompromised host the more frequent pathogens were: enteroaggregative E.coli (19,6%), Microsporidium(9.4%), Cryptosporidium sp.(8.2%), Rotavirus (8.2%), Shigella sp. (7.6%), Candida albicans (7.6%), Entamoeba histolytica (5,1%), Salmonella enterica (4.4%), Isospora belli (4.4%) and Blastocystis hominis (2,5%). Isolated enteropathogenic strains displayed high resistance to most antibiotics used in Senegal for treating diarrhea (tetracycline, cotrimoxazole); they were susceptible to quinolons and cephalosporins III .

Etiologies infectieuses des diarrhees de l'adulte au cours de l'infection a VIH a Dakar: etude cas / temoins sur 594 maladse

Cette etude prospective sur 594 patients a ete menee a Dakar (Senegal) de mai 1997 a mai 1999 sur la diarrhee au cours du sida.Les 594 malades de l'etude cas / temoins ont ete repartis en 4 groupes: les cas constitues par les groupes I (D+VIH+ et II (D+VIH-)et les temoins par les groupes III (D-VIH+) et IV (D-VIH-).Les pathogenes stricts ont ete identifies par les techniques conventionnelles; les parasites opportunistes ont ete mis en evidence par les techniques de coloration de Kinyoum et du trichome de Weber; la recherche du rotavirus a ete realisee par une technique d'agglutination sur carte.Quant a l'etude de la sensibilite; elle a ete realisee par la technique de diffusion en milieu gelose. L'etude de la sensibilite aux antibiotiques des germes anteropathogenes isoles a montre un haut niveau de resistance aux antibiotiques les plus souvent prescrits au Senegal pour le traitement de la diarrhee (cotrimoxazole; tetracyclines). L'antibiotherapie probabiliste de premier choix devrait faire appel aux fluoroguionolones qui offrent parmi toutes les molecules actives sur les bacteries isolees le rapport cout / efficacite le plus interessant

[The role of integrons in dissemination of antibiotic resistance]. / Place des intégrons dans la dissémination de la résistance aux antibiotiques.

Bacteria can transfer genetic information to get protection against most antibiotics. The acquisition of resistance genes involves genetic mobile elements such as plasmids and transposons. Another genetic structures, named integrons, have been described and contain one or more gene cassettes located at a specific site. Integrons contain an intI gene encoding a site-specific recombinase belonging to the integrase family and a recombination site attI. A gene cassette includes an open reading frame and, at the 3'-end, a recombination site attC. Integration or excision of cassettes occurs by a site-specific recombination mechanism catalyzed by the integrase. However, insertion can rarely occur, at non-specific sites leading to a stable situation for the cassette. Cassettes are transcribed from a common promoter located in the 5'-conserved segment and expression of distal genes is reduced by the presence of upstream cassettes. Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described. Integrons seem to have a major role in the spread of multidrug resistance in Gram-negative bacteria but integrons in Gram-positive bacteria have been recently described. Moreover, the finding of super-integrons with gene cassettes coding for other determinants (biochemical functions, virulence factors) in different Gram negative bacteria suggests that integrons are probably implied in bacterial genome evolution.

[Micromethod for identification of enterobacteria]. / Microméthodes d'identification des Enterobactéries.

The aim of this study was to set accurate and reliable methods in the identification of Enterobacteriaceae. In Micro CSB Entero each of a strip with 20 cupules containing dehydrated substrates for biochemical identification of bacterial species. Strips were inoculated with 100 microliters per cupule and incubated. After 18 hours, reagents were added. Baye's theorem was used to validate tests. Reactions from Micro CSB were cleared and easily read. 102 strains of Enterobacteriaceae were identified by MicroCSB--89.3% were correctly identified. 0.9% were identified with other tests 9.8% were incorrectly identified MicroCSB gives many advantages: This method save time, space and is less expensive than the other methods and it gives security for identifying species belonging to the different genera.

[Micromethod for the identification of streptococcal, enterococcal and staphylococcal species]. / Microméthodes d'identification des Streptocoques, Enterocoques et des Staphylocoques.

The aim of this study was to set accurate and reliable methods in the identification of streptococcal, enterococcal and staphylococcal species. Micro CSB Strep and Staph system consists each of a strip with cupules containing dehydrated substrates for biochemical identification of bacterial species. Baye's theorem was used to validate tests. Reactions from micromethods were clear and easily read. Identification of 229 strains of streptococci and enterococci was correct for most species with 98.7% species with 99.3% sensitivity. 41 strains of staphylococci were also correctly identified with 85.2% of specificity and 97.68% of sensitivity.

Phenotypic and genotypic characterization of Vibrio cholerae isolates from a recent cholera outbreak in Senegal: comparison with isolates from Guinea-Bissau.

A total of 127 strains of Vibrio cholerae (117 V. cholerae O1 and 10 nonagglutinating strains) isolated from a recent cholera outbreak in Senegal and four strains isolated in Guinea-Bissau (during the survey of a cholera epidemic that occurred 10 months before the Senegalese one) were analyzed. Strains were characterized by conventional methods (biochemical and serologic identification, susceptibility to antimicrobial agents), polymerase chain reaction for genes encoding cholera toxin (CtxA), zonula occludens toxin (Zot), and accessory cholera enterotoxin (Ace), and by ribotyping. Conventional methods showed that all strains of V. cholerae O1 belonged to serotype Ogawa, biotype El Tor and were resistant to the vibriostatic agent O129 (2,4-diamino 6,7-diisopropylpteridine phosphate), cotrimoxazole, and chloramphenicol; all strains were sensitive to tetracycline, a drug that has been extensively used in cholera therapy. Most of these V. cholerae O1 (112 strains from Senegal and four strains from Guinea-Bissau) had an intact core region (virulence cassette) and amplified a 564-basepair (bp) fragment of ctxA, a 1083-bp fragment of zot, and a 314-bp fragment of ace. Ribotyping of V. cholerae O1 strains after Bgl I restriction of total DNA revealed that ribotype B5a, which is the predominant ribotype of this seventh pandemic of cholera, was not isolated. Instead, a new ribotype was identified and designated B27 in our data bank. Since O1 isolates from Guinea-Bissau and Senegal have the same biotype, serotype, and ribotype and as the Guinea-Bissau outbreak that preceded the one in Senegal, this emerging ribotype probably came from Guinea-Bissau. Nonagglutinating strains exhibited no resistance to the O129 agent and to the tested antibiotics, they were all negative for virulence cassette, except for one strain with the ctxA and zot genes isolated from a patient with diarrhea, and there was a great variability of ribotypes among these strains. There was no difference between environmental O1 strains isolated from water and strains isolated from patients with cholera, suggesting that fecally contaminated water is an important reservoir for infection.

Guinea pig intestinal phospholipase B: protein expression during enterocyte maturation and effects of N-oligosaccharide removal on enzymatic activities and protein stability.

Guinea pig phospholipase B (PLB) is an intestinal brush-border hydrolase displaying a broad substrate specificity towards various dietary lipids. PLB was detected by immunoblotting as a single 140-kDa polypeptide in all cell populations isolated from guinea pig intestinal mucosa, but increased in parallel to its activity from undifferentiated to mature cells, the specific activity of the enzyme remaining constant. Moreover, N-glycosylation, which contributed to 23% of the apparent molecular mass, was identical along the cell differentiation axis. In all cell fractions, N-linked sugar chains were of the complex type, since they were removed by N-glycosidase F, whereas PLB remained insensitive to endoglycosidase H. Moreover, lack of O-glycosylation was demonstrated by the insensitivity of PLB to O-glycosidase and by its failure to interact with Helix pomatia lectin after prior treatment with neuraminidase or alpha-fucosidase. Enzymatic removal of sugar chains reduced phospholipase A2, lysophospholipase and diacylglycerol lipase activities by 27-35%, kinetic analysis indicating a decrease in apparent Vmax values for the three enzymatic activities, whereas the Km remained unchanged. Finally, the carbohydrate-depleted form of PLB did not display gross changes in thermal stability, in contrast to PLB from microorganisms previously investigated. Our data indicate that the high level of PLB N-glycosylation is poorly related to its biological function. Whether carbohydrate chains are involved in proper targeting of the enzyme to the brush-border membrane remains to be established.