Preliminary results on a proposed histopathological assessment of predictive factors for basal cell carcinoma recurrence after primary free margin excision.

Background: Basal cell carcinoma (BCC) incidence is steadily increasing but therapeutic solutions remain limited and present a public health challenge. Aims: To identify predictive factors of BCC recurrence after primary free margin excision, with automated methods, by evaluating cell proliferation, the Hedgehog pathway activation and primary cilia. Materials and Methods: This case-control study included 32 patients (16 with recurrence occurring at least 6 months after complete resection, and 16 without recurrence) who underwent surgery for BCC. Formalin-fixed paraffin-embedded cutaneous resections were processed for immunohistochemistry or immunostaining with the following primary antibodies: mouse anti-MCM6, rabbit anti-ARL13B and rabbit anti-GLI1. Results: BCC recurrence after free margin excision was significantly linked to a higher proliferative index (p < 0.001) and a lower cilia count (p = 0.041) in the primary lesion. No significant differences were observed regarding cilia length (p = 0.39) or GLI1-positive nuclei. Discussion: The complex interplay between essential signaling pathways, cell proliferation and cilia requires further experimental investigations in the context of BCC recurrence. Conclusion: A higher proliferative index evaluated with MCM6 antibody could be a useful prognosis marker of BCC risk of recurrence. The lower cilia count in the primary lesion unveiled novel perspectives to understand BCC recurrence molecular mechanisms.

Outcome and molecular landscape of patients with PIK3CA-mutated metastatic breast cancer.

BACKGROUND: α-Selective phosphatidylinositol 3-kinase (PI3K) inhibitors improve outcome in patients with PIK3CA-mutated, hormone receptor-positive (HR+)/Her2- metastatic breast cancer (mBC). Nevertheless, it is still unclear how to integrate this new drug family in the treatment landscape. PATIENTS AND METHODS: A total of 649 patients with mBC from the SAFIR02 trial (NCT02299999), with available mutational profiles were selected for outcome analysis. PIK3CA mutations were prospectively determined by next-generation sequencing on metastatic samples. The mutational landscape of PIK3CA-mutated mBC was assessed by whole-exome sequencing (n = 617). Finally, the prognostic value of PIK3CA mutations during chemotherapy was assessed in plasma samples (n = 44) by next-generation sequencing and digital PCR. RESULTS: Some 28% (104/364) of HR+/Her2- tumors and 10% (27/255) of triple-negative breast cancer (TNBC) presented a PIK3CA mutation (P < 0.001). PIK3CA-mutated HR+/Her2- mBC was less sensitive to chemotherapy [adjusted odds ratio: 0.40; 95% confidence interval (0.22-0.71); P = 0.002], and presented a worse overall survival (OS) compared with PIK3CA wild-type [adjusted hazard ratio: 1.44; 95% confidence interval (1.02-2.03); P = 0.04]. PIK3CA-mutated HR+/Her2- mBC was enriched in MAP3K1 mutations (15% versus 5%, P = 0.0005). In metastatic TNBC (mTNBC), the median OS in patients with PIK3CA mutation was 24 versus 14 months for PIK3CA wild-type (P = 0.03). We further looked at the distribution of PIK3CA mutation in mTNBC according to HR expression on the primary tumor. Some 6% (9/138) of patients without HR expression on the primary and 36% (14/39) of patients with HR+ on the primary presented PIK3CA mutation (P < 0.001). The level of residual PIK3CA mutations in plasma after one to three cycles of chemotherapy was associated with a poor OS [continuous variable, hazard ratio: 1.03, 95% confidence interval (1.01-1.05), P = 0.007]. CONCLUSION: PIK3CA-mutated HR+/Her2- mBC patients present a poor outcome and resistance to chemotherapy. Patients with PIK3CA-mutated TNBC present a better OS. This could be explained by an enrichment of PIK3CA mutations in luminal BC which lost HR expression in the metastatic setting. TRIAL REGISTRATION: SAFIR02 trial: NCT02299999.

The Blomia tropicalis allergen Blo t 7 stimulates innate immune signalling pathways through TLR2.

BACKGROUND: Although the house dust mite species Blomia tropicalis is a leading cause of allergic diseases in tropical and subtropical regions, the identification and characterization of the allergenic proteins remain incomplete. OBJECTIVE: We aimed to characterize a recombinant form of Blo t 7 (rBlo t 7) in terms of IgE reactivity, lipid-binding activity and ability to stimulate innate immunity. METHODS: The mature Blo t 7 cDNA was cloned by PCR methods for the expression of a secreted form of the allergen in P. pastoris. The IgE reactivity to purified rBlo t 7 as well as the potential cross-reactivity with Der p 7 was determined by ELISA. The lipid-binding capacity of rBlo t 7 was assayed using fluorescent lipid probes. The stimulation of TLR2 signalling pathway by rBlo t 7 was examined in cell activation and reporter assays. RESULTS: The amplified mature Blo t 7 cDNA revealed the presence of a 60 base pair insertion compared with the reference sequence registered in the GenBank database. Multiple protein sequence alignments of group 7 mite allergens confirmed that this longer deduced amino acid sequence was the authentic Blo t 7 polypeptide chain. Analysis of IgE reactivity can classify rBlo t 7 as an intermediate B. tropicalis allergen which displayed weak cross-reactivity with Der p 7. Purified rBlo t 7 was shown to bind selectively the naturally fluorescent lipid probe cis-parinaric (cPNA) with a dissociation constant of 2 µmol/L. The group 7 Blomia allergen stimulated the TLR2-, NF-kB- and MAPK-dependent production of IL-8 and GM-CSF in respiratory epithelial cells. CONCLUSIONS & CLINICAL RELEVANCE: Through its propensity to transport fatty acids/lipids and to stimulate TLR2 signalling pathways in airway epithelial cells, Blo t 7 can represent a key allergen for the initiation of the B. tropicalis-induced airway inflammation.

[Quilting suture after mastectomy in prevention of postoperative seroma: a prospective observational study]. / Intérêt du capitonnage de la loge de mastectomie dans la prévention des séromes post-opératoires : étude prospective.

OBJECTIVES: The occurrence of a postoperative seroma is the main complication of mastectomy. In 2011, Ouldamer et al. adapted a quilting technique used in reconstructive surgery in mastectomy closure. The aim of this study is to evaluate the impact of quilting in the prevention of postoperative seroma. PATIENTS AND METHODS: This is an observational prospective study to the Centre Hospital-University of Tours. Hundred and forty-four patients who underwent a mastectomy between January 1st, 2011 and October 1st, 2012 were included. Patients were divided into 2 groups, one with a classic wound closure with drainage and the second with quilting suture of skin flaps to the underlying musculature after mastectomy without drainage. RESULTS: Quilting suture significantly reduces the postoperative seroma appearance (OR=0.15; CI95% [0.06-0.39]; P<0.001). Operative time is increased by 20minutes in the quilted group (P<0.001). Postoperative pain is not changed by quilting. The duration of hospitalization is significantly shorter (5.09±1.46 days versus 6.49±2.77 days; P<0.001). Quality of the healing and appearance of the scar, rated by patients, are identical in both groups. CONCLUSION: Quilting is an effective method not only for prevention of seroma, but also for reducing of hospitalization duration, without increasing of postoperative pain and complications.

Development of recombinant stable house dust mite allergen Der p 3 molecules for component-resolved diagnosis and specific immunotherapy.

BACKGROUND: The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the diagnosis of allergy and the safety and efficacy of immunotherapy. OBJECTIVE: The current study reports the immunological characterization of two recombinant molecules of the HDM allergen Der p 3 as useful tools for diagnosis and immunotherapy. METHODS: Recombinant mature (rDer p 3) and immature (proDer p 3) Der p 3 and their corresponding S196A mutants were produced in Pichia pastoris and purified. The stability, IgE-binding capacity and allergenicity of the different proteins were analysed and compared with those of the major mite allergen Der p 1 used as a reference. Additionally, the immunogenicity of the different allergens was evaluated in a murine model of Der p 3 sensitization. RESULTS: Compared to the IgE reactivity to recombinant and natural Der p 3 (nDer p 3), the mean IgE binding of patient's sera to rDer p 3-S196A (50%) was higher. The poorly binding to nDer p 3 or rDer p 3 was due to autolysis of the allergen. Contrary to Der p 3, proDer p 3 displayed very weak IgE reactivity, as measured by sandwich ELISA and competitive inhibition, rat basophil leukaemia degranulation and human basophil activation assays. Moreover, proDer p 3 induced a TH 1-biased immune response that prevented allergic response in mice but retained Der p 3-specific T-cell reactivity. CONCLUSION: rDer p 3-S196A should be used for the diagnosis of HDM allergy elicited by Der p 3, and proDer p 3 may represent a hypoallergen of Der p 3.

[Profits of post-menopausal ovarian conservation at the time of hysterectomy for benign disease: mirage or reality?]. / Bénéfices de la conservation ovarienne post ménopausique lors d'une hystérectomie pour pathologie bénigne : mirage ou réalité ?

BACKGROUND: This study aimed to summarize the existing literature on the benefice-risk balance for ovarian conservation at the time of hysterectomy for benign disease in post menopausal women not at high risk for ovarian cancer. METHODS: We searched the published English and French literature using search engines from PUBMED, Medline for studies related to outcomes following hysterectomy with bilateral oophorectomy for benign disease and risk-reducing surgery for ovarian cancer. RESULTS: There are in the literature arguments to support systematic bilateral oophorectomy in post menopausal women not at high risk for ovarian cancer (prevention of ovarian cancer, ovarian benign disease and chronic pelvic pain due to postoperative ovarian adhesions). There are also arguments against postmenopausal oophorectomy (effect on endocrine function, bone density, cardiovascular disease and increased mortality). Before the age of 65, there is no formal argument allowing to recommend an attitude rather than another. On the other hand, beyond the age of 65 years, the literature is clear and a bilateral salpingooophorectomy is recommended. CONCLUSION: Before the age of 65 years, benefits and relative risks of bilateral oophorectomy at the time of hysterectomy for benign disease even in post menopausal patients should be considered on an individual basis by clinicians and patients. Beyond the age of 65 years, the literature is clear and a bilateral salpingooophorectomy is recommended.

[Pelvic actinomycosis: just think of it]. / Actinomycose pelvienne : est-ce prévisible ?

OBJECTIVES: Pelvic actinomycosis is a rare disease that can be diagnosed before, during or after surgical treatment of a suspected ovarian tumor, a suspected bowel obstruction, or acute peritonitis. The possibility of early detection of pelvic or abdominal abscess related to was evaluated through a personal series and literature review. PATIENTS AND METHODS: Our series of 11 cases of severe abdominal or pelvic actinomycosis is related and compared to 58 cases reported in the literature. RESULTS: Seven patients in this series were diagnosed with pelvic inflammatory disease and acute peritonitis with or without bowel obstruction, and four women were diagnosed after surgical treatment for suspected ovarian cancer. Fifty-two of the 58 cases of reproductive tract actinomycosis reported in the literature review and all our cases were associated with prolonged use of an intrauterine contraceptive device with a mean of eight years. The contribution of pelvic ultrasound and angioscanner in evaluating these patients should not be underestimated and MRI may be useful in some cases as well. Early diagnosis based on Actinomyces-positive cervical smears or abscess aspiration was accomplished only once in our series and was rare in literature. A histopathologic diagnosis during laparoscopy or laparotomy could avoid more difficult and extensive surgery. In our series of 11 patients, five women required abdominal surgery, five required salpingo-oophorectomy and three required hysterectomy. All women required surgical intervention. Effective treatment combined long antibiotic therapy with surgery. Correct preoperative diagnosis is rare but if achieved, long-term treatment with penicillin for at least two months and sometimes up to a year may completely eradicate the infection. Surgery may still be necessary to improve medical treatment or to resolve pelvic abscesses. DISCUSSION AND CONCLUSION: Any pelvic abscess occurring in a woman with a history of long-term use of an intrauterine device should be considered as possible pelvic actinomycosis. If there is no fever in association with an atypical adnexal tumor, frozen section should be obtained during surgery to rule out the diagnosis of actinomycosis.

Production and immunogenicity of hypoallergenic codon-optimized DNA vaccine encoding mature Der p 1 allergen.

BACKGROUND: Genetic vaccination with plasmid DNA encoding allergens is a promising potential approach for the treatment or prevention of allergy. Nonetheless, because the allergens expressed can display immunoglobulin (Ig) E reactivity, methods to deliver hypoallergenic variants can minimize the risk of type 2 helper (T(H)2) cell priming after DNA immunization. METHODS: A humanized synthetic gene encoding mature Dermatophagoides pteronyssinus group 1 (Der p 1) allergen was cloned into the pHIS expression vector carrying unmethylated CpG 2006 (CpG 2006) motif but devoid of signal sequence. The immunogenicity of this DNA construct was compared in naïve mice with that of recombinant ProDer p 1 protein adjuvanted with alum. RESULTS: Codon optimization of the cDNA encoding mature Der p 1 markedly improved allergen expression. Mature Der p 1, expressed intracellularly in Human Embryonic Kidney 293 cells (HEK 293 cells) transfected with codon-optimized Der p 1 cDNA (pHIS-mHuDer p 1), was shown to be hypoallergenic as it displayed no IgE reactivity. Intradermal vaccinations of naïve Balb/C mice with pHIS-mHuDer p 1 elicited an allergen-specific T(H)1 response characterized by the production of specific IgG2a, a very low amount of specific IgG1, and no specific IgE. Lipoplex formulation with cationic liposome composed of lecithin, N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) and cholesterol not only accelerated the induction of T(H)1 response but also increased its intensity. CONCLUSION: A codon-optimized DNA vaccine encoding mature Der p 1 in a lipoplex formulation could represent a promising hypoallergenic vaccine candidate for safer immunotherapy against house dust mite allergy.

Immunomodulatory properties of Lactobacillus plantarum and its use as a recombinant vaccine against mite allergy.

BACKGROUND: Selected lactic acid bacteria were reported to prevent atopic dermatitis and experimental asthma but the mechanisms of their immunomodulatory effects are not fully elucidated. In this study, the signaling pathways triggered by Lactobacillus plantarum NCIMB8826 were investigated and the potential use of this strain producing a variant of the mite allergen Der p 1 as live vaccine vehicle was evaluated. METHODS: Mouse bone marrow-derived dendritic cells were stimulated with wild-type or a L. plantarum teichoic acid mutant to evaluate the secretion of cytokines. A recombinant L. plantarum expressing Der p 1 was engineered, its in vitro immunomodulatory properties were characterized and its prophylactic potential was evaluated in a Der p 1-sensitization murine model. RESULTS: Mouse dendritic cells stimulated by L. plantarum triggered the release of interleukin-10 (IL-10), IL-12 p40, IL-12 p70 and tumor necrosis factor-alpha (TNF-alpha). IL-12 p40 secretion was dependent on nuclear factor-kappaB (NF-kappaB), mitogen-activated protein (MAP) kinases, Toll-like receptor 2 (TLR2), TLR9 and on the bacterial teichoic acid composition. Recombinant L. plantarum producing Der p 1 exhibited similar immunostimulatory properties as wild-type. Prophylactic intranasal pretreatment of mice with this recombinant strain prevented the development of the typical Th2-biased allergic response by a drastic reduction of specific IgE and the induction of protective allergen-specific IgG2a antibodies. Moreover, both wild-type or recombinant L. plantarum reduced airway eosinophilia following aerosolized allergen exposure and IL-5 secretion upon allergen restimulation. CONCLUSION: By combining both Th1-type immunostimulatory properties and an efficient allergen delivery capacity, recombinant L. plantarum producing Der p 1 represents a promising vaccine against house dust mite allergy.

In vivo and in vitro immunomodulation of Der p 1 allergen-specific response by Lactobacillus plantarum bacteria.

BACKGROUND: Lactic acid bacteria (LAB) were reported to reduce some allergic manifestations in mice and humans but their impact on the aeroallergen-dependent immune mechanisms is still debated. OBJECTIVE: The potential capacities of Lactobacillus plantarum NCIMB8826 to reduce the allergic response induced by Der p 1, the major house dust mite allergen of Dermatophagoides pteronyssinus, were evaluated in vivo and in vitro. Methods First, the effect of the intranasal co-administration of LAB and purified Der p 1 allergen before a sensitization protocol was evaluated. The allergen-specific antibody and cellular responses as well as airway inflammation were measured. Second, the impact of LAB on the cytokine profile of spleens cells from Der p 1-sensitized mice was assessed. Third, upon stimulation with LAB, the levels of cytokine produced by dendritic cells derived from the bone marrow (BMDCs) of wild-type, Toll-like receptor 2 (TLR2)-, TLR4- and MyD88-KO mice were compared. Results The co-application of L. plantarum and Der p 1 induced a T-helper type 1 (Th1)-biased allergen-specific IgG response, the absence of specific IgE response and favoured the production of INF-gamma upon allergen re-stimulation. Moreover, the previous LAB administration reduced the development of bronchoalveolar lavage eosinophilia usually induced by aerosol exposure. Additionally, the studied LAB strain was shown to modify in vitro the cytokine level produced by Der p 1-sensitized spleen cells mainly towards a Th1 profile. Finally, L. plantarum stimulated high IL-12 and moderate IL-10 production in mouse BMDCs notably through the TLR2-, MyD88-dependent and TLR4-independent pathway. CONCLUSION: In vivo co-administration of probiotic LAB with Der p 1 might prevent the development of the mite allergic response. The probiotic L. plantarum was shown to display in vitro therapeutic potentials for the treatment of allergy and to trigger the immune system by a TLR2- and MyD88-dependent signalling pathway.

Absence of immunoglobulin E synthesis and airway eosinophilia by vaccination with plasmid DNA encoding ProDer p 1.

BACKGROUND: Various studies have shown that immunization with naked DNA encoding allergens induces T helper 1(Th1)-biased non-allergic responses. OBJECTIVE: To evaluate the polarization of the immune responses induced by vaccinations with plasmid DNA encoding the major mite allergen precursor ProDer p 1. METHODS: A DNA vaccine was constructed on the basis of a synthetic cDNA encoding ProDer p 1 with optimized codon usage. The immunogenicity of ProDer p 1 DNA in CBA/J mice was compared with that of purified natural Der p 1 or recombinant ProDer p 1 adjuvanted with alum. Vaccinated mice were subsequently exposed to aerosolized house dust mite extracts to provoke airway inflammation. The presence of inflammatory cells was examined in bronchoalveolar lavage (BAL) fluids and allergen-specific T cell reactivity was measured. RESULTS: Naive mice immunized with ProDer p 1 DNA developed Th1 immune responses characterized by high titres of specific IgG2a antibodies, low titres of specific IgG1 and, remarkably, the absence of anti-ProDer p 1 IgE. No specific responses were observed in animals vaccinated with the blank DNA vector. By contrast, natural Der p 1 or recombinant ProDer p 1 adsorbed to alum induced pronounced Th2 allergic responses with strong specific IgG1 and IgE titres. Spleen cells from DNA ProDer p 1-vaccinated mice secreted high levels of IFN-gamma and low production of IL-5. Conversely, both adjuvanted allergens stimulated typical Th2-type cytokine profile characterized by high and low levels of IL-5 and IFN-gamma, respectively. Whereas BAL eosinophilia was clearly observed in Der p 1-immunized animals, ProDer p 1 DNA as well as ProDer p 1 vaccinations prevented airway eosinophil infiltrations. CONCLUSIONS: These results suggest that vaccination with DNA encoding ProDer p 1 effectively fails to induce the allergen-induced IgE synthesis and airway cell infiltration. Plasmid DNA encoding ProDer p 1 may provide a novel approach for the treatment of house dust mite allergy.

High-level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris.

BACKGROUND: The major house dust mite Der p 1 allergen is associated with allergic disease. Heterologous over-expression of biologically active Der p 1 was previously attempted but with limited success. OBJECTIVE: The aim of this study was to establish an efficient system for the production of recombinant Der p 1. METHODS: The proform of Der p 1 was expressed in Pichia pastoris as a fusion with the alpha mating factor signal sequence. The recombinant product was purified from culture medium and compared to Der p 1 isolated from mite culture, in terms of enzymatic activity as well as IgE binding capacity. RESULTS: ProDer p 1 was efficiently secreted into culture medium as a hyperglycosylated protein of 40-60 kDa. Postpurification dialysis in acidic buffer was required for the autocatalytic processing of Der p 1. During this treatment, the prosequence was efficiently removed to give highly glycosylated recombinant mature Der p 1. Competition ELISA experiments as well as cysteine proteinase activity assays indicated that recombinant processed Der p 1 was similar to natural Der p 1 isolated from mite cultures in terms of IgE binding and enzymatic activities. However, the histamine releasing activity of recombinant Der p 1 was slightly weaker than that of natural Der p 1. CONCLUSION: This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.

Th2 polarization by Der p 1--pulsed monocyte-derived dendritic cells is due to the allergic status of the donors.

The polarization of the immune response toward a Th2 or a Th1 profile can be mediated by dendritic cells (DCs) following antigen presentation and interaction with T cells. Costimulatory molecules such as CD80 and CD86 expressed by DCs, the polarizing cytokine environment during DC--T-cell interaction, and also the nature of the antigen are critical in the orientation of the immune response. In this study, the effect of the cysteine protease Der p 1, one of the major allergens of the house dust mite Dermatophagoides pteronyssinus, on these different parameters was evaluated comparatively on monocyte-derived DCs obtained from healthy donors, from pollen-sensitive patients, or from patients sensitive to Dermatophagoides pteronyssinus. Results showed that Der p 1 induced an increase in CD86 expression only on DCs from house dust mite--sensitive patients. This was also associated with a higher capacity to induce T-cell proliferation, a rapid increase in the production of proinflammatory cytokines, tumor necrosis factor--alpha and interleukin (IL)-1 beta, and the type 2 cytokine IL-10. No changes in the release of IL-12 p70 were induced by Der p 1. Finally, purified T cells from house dust mite-sensitive patients stimulated by autologous Der p 1--pulsed DCs preferentially produced IL-4 rather than interferon-gamma. These effects were abolished in the presence of the inactive precursor of Der p 1 (ProDer p 1). Taken together, these data suggest that DCs from house dust mite--sensitive patients, in contrast to DCs from healthy donors and from pollen-sensitive patients, exposed to Der p 1 play a pivotal role in the enhancement of the Th2 response associated with the allergic reaction developed in response to house dust mite exposure. (Blood. 2001;98:1135-1141)

Diet acids and alkalis influence calcium retention in bone.

The urine-acidifying properties of food constituents depend on their content of non-oxidizable acids or precursors. Acidifying constituents such as animal proteins may negatively affect calcium metabolism and accelerate bone resorption, thus representing an aggravating factor for osteoporosis. This four-period, double-crossover study investigated whether a diet intervention specifically focused on acid load could modify calcium metabolism in humans. Eight healthy volunteers underwent a four-day metabolic preparation with two types of diets, one rich in acid ash-forming nutrients, and one providing base-forming nutrients (including bicarbonate-rich mineral water), both having similar contents of calcium, phosphate, sodium, proteins and calories. On the fourth day, a single oral dose of 1 g calcium was given, either as carbonate or as gluconolactate. Serial blood and urine samples revealed that the diet affected blood pH (average difference 0.014, p=0.002) and urine pH (average difference 1.02, p<0.0001) in the expected direction, but had no influence on the absorption of the calcium supplement. The acid-forming diet increased urinary calcium excretion by 74% when compared with the base-forming diet (p<0.0001), both at baseline and after the oral calcium load, and C-telopeptide excretion by 19% (p=0.01), suggesting a skeletal origin for the excess calcium output. This observation confirms that renally excreted acids derived from food influence calcium metabolism, and that alkalizing nutrients inhibit bone resorption. Further studies are needed to determine the clinical impact of dietary counseling for avoiding diet acids as a preventive measure against osteoporosis.

Cloning, expression and purification of recombinant cotton rat interleukin-5.

The coding sequence of the hispid cotton rat (Sigmodon hispidus) interleukin-5 (IL-5) was isolated by a combination of reverse transcription (RT)-PCR and RACE protocols from concanavalin A stimulated spleen cells. The open reading frame of 399 bp encodes a polypeptide of 132 amino acids. Comparison with the rat, mouse, gerbil and human counterparts revealed 88, 88, 87 and 75% identity at the nucleotide level and 88, 90, 89 and 70% at the amino acid level, respectively. The entire coding sequence, minus the putative signal peptide sequence, was inserted into an inducible Escherichia coli expression vector. The recombinant protein possessed an expected molecular mass of 14kDa and was located in bacterial inclusion bodies. A purification scheme under reducing and denaturing conditions followed by subsequent successive dialysis steps led to the recovery of a recombinant dimeric cotton rat IL-5. The biological activity of the recombinant protein was demonstrated in a murine cell line proliferation assay. This activity was specifically inhibited by rat monoclonal antibodies directed against mouse IL-5. Together with specific antibodies that can be generated easily, cotton rat IL-5 constitutes a useful tool for extending the use of the cotton rat animal model in the study of various human pathogens.

Differential neutralizing antibody responses to varicella-zoster virus glycoproteins B and E following naked DNA immunization.

The only available vaccine against varicella-zoster virus (VZV) consists of the VZV-Oka attenuated but persistent virus strain. Development of a safer, subunit vaccine is therefore desirable. In this prospect, nucleic acid vaccines, expressing truncated forms of VZV glycoproteins B (recgB) and E (recgE) from which the anchor and the cytoplasmic domains were deleted, were used to immunize mice. Vaccination with recgB encoding plasmid elicited a strong and specific humoral immune response. Total IgG and neutralizing titres were comparable to those previously obtained by vaccination with purified and adjuvanted native recgB. In contrast, mice immunization with recgE encoding plasmid only induced a very weak immune response whereas we previously showed that vaccination with adjuvanted native or denatured recgE protein led to high neutralizing titres. The weakness of the immune response induced by recgE-encoding plasmid depended neither on the deletion of the anchor domain in the gE gene nor on the animal model. Analysis of antibody isotypes produced by plasmid immunizations revealed a response slightly dominated by IgG2a. Taken together, the data indicate that a VZV subunit vaccine based on adjuvanted recombinant glycoprotein E is more promising than a nucleic acid-based vaccine strategy. As regards recgB, both vaccination approaches might be appropriate.

Susceptibility to levofloxacin of clinical isolates of bacteria from intensive care and haematology/oncology patients in Switzerland: a multicentre study.

The objective of this study was to examine the susceptibility of clinical isolates to levofloxacin, a fluoroquinolone with extended activity against Gram-positive bacteria, and other antibiotics in 12 Swiss clinical microbiology laboratories using the NCCLS disc diffusion technique. Isolates were prospectively collected from intensive care units (ICUs (59%), oncology wards (7%) and other units with haematology/oncology patients (34%) from June 1995 to March 1996. The levofloxacin breakpoints used were as recommended by the manufacturer. A total of 310 Gram-positive and 580 Gram-negative isolates from the respiratory tract (36%), skin/wounds (12%), blood (16%), urine (17%) and other sources (19%) were tested. The percentage of isolates susceptible to levofloxacin was 100% for Enterococcus spp. (38 strains), Streptococcus agalactiae (13), Streptococcus pneumoniae (65), Acinetobacter spp. (11), Citrobacter diversus (6), Citrobacter freundii (17), Klebsiella oxytoca (39), Morganella morganii (16), Proteus mirabilis (20), Proteus vulgaris (23), Serratia spp. (19), Stenotrophomonas maltophilia (10) and Haemophilus influenzae (41). The percentage of isolates susceptible to levofloxacin for Staphylococcus aureus (95 strains, including 2% MRSA) was 94%, coagulase-negative staphylococci (85) 65%, Enterobacter spp. (75) 99%, Escherichia coli (111) 97%, Klebsiella pneumoniae (45) 98% and Pseudomonas aeruginosa (124) 87%. In conclusion, levofloxacin is a new fluoroquinolone to which the most common clinical isolates in Switzerland are susceptible. The susceptibility of Enterococcus spp. and S. pneumoniae to levofloxacin was particularly remarkable. This compound appears to be a promising therapeutic alternative for the treatment of Gram-positive infections.

The varicella zoster virus glycoprotein B (gB) plays a role in virus binding to cell surface heparan sulfate proteoglycans.

Varicella-zoster virus (VZV) interacts with cell surface heparan sulfate proteoglycans during virus attachment. In the present study, we investigated the potential involvement of two VZV glycoproteins, gB and gE, in the virus adsorption process. We showed that gB, but not gE, binds specifically to cellular heparan sulfate proteoglycans (HSPGs). Indeed, soluble recombinant gB protein (recgB) was found to bind to immobilized heparin and to MRC5 and L cells, a binding which was inhibited by heparin. Furthermore, recgB binding to two heparan sulfate-minus mutant L cell lines, gro2C and sog9 cells, was markedly reduced as compared to the parental L strain. Under the same experimental conditions, soluble recombinant VZV gE protein did not interact with heparin or with cell surfaces. We also demonstrated that the gB-HSPGs interactions were relevant to the VZV attachment to cells. Indeed, although polyclonal antibodies directed to gB did not impair the VZV binding, recgB could delay the virus adsorption. Our results thus strongly suggest that the interactions between gB and heparan sulfate proteoglycans take part in the initial VZV attachment to cell surfaces.

Neutralizing antibody responses induced by varicella-zoster virus gE and gB glycoproteins following infection, reactivation or immunization.

The purpose of this study was to compare the antibody responses to varicella-zoster virus (VZV) gE and gB after natural VZV infection and after vaccination with live attenuated OKA vaccine in order to determine the relative importance of these proteins as components of a subunit vaccine. Anti-VZV antibody titers determined by IFA were of the same order of magnitude in sera from individuals with a history of varicella and in vaccinated children but higher in individuals given booster vaccination. The titers of anti-gE and anti-gB antibodies were measured by ELISA using recombinant gE or gB as capture antigen. From these experiments, it appears that the ratio of anti-gE to anti-gB antibody is highly variable from one individual to another but relatively stable over a long period of time for a particular individual, even after a zoster episode. Neutralizing antibodies directed against gE or gB were also measured by subtracting the neutralization titers obtained before and after depletion of the specific antibodies on immobilized recombinant gE, gB, or both. This showed that, with respect to neutralization, anti-gE and anti-gB are equally prevalent in vaccinated children and that anti-gE is generally, but not always, predominant over anti-gB in VZV-infected individuals. Finally, antibodies to these two glycoproteins appear to be predominant among the neutralizing antibodies directed to other VZV antigens.