Neisseria meningitidis expressing transferrin binding proteins of Actinobacillus pleuropneumoniae can utilize porcine transferrin for growth

Homologous recombination was used to generate a number of mutants of serogroup B Neisseria meningitidis B16B6 with the following characteristics: (i) an inability to bind human or porcine transferrin because of loss of both transferrin binding proteins (Tbp) A and B [strain B16B6(Str(r))/tbpA(-)B(-)] and (ii) an ability to bind porcine transferrin but not human transferrin [strain B16B6(Str(r))/tbpA(ap)B(ap)] due to replacement of the meningococcal Tbp with the Tbp of Actinobacillus pleuropneumoniae. During construction of the B16B6(Str(r))/tbpA(ap)B(ap) strain, transformants expressing only TbpA or TbpB of A. pleuropneumoniae were isolated [strains B16B6(Str(r))/tbpA(ap)B(-) and B16B6(Str(r))/tbpA(-)B(ap)]. Expression of the A. pleuropneumoniae Tbp in N. meningitidis B16B6 was iron regulated and expressed under the control of the meningococcal promoter. The relative abilities of the meningococcal transformants to bind porcine transferrin were in the order B16B6(Str(r))/tbpA(ap)B(ap) > B16B6(Str(r))/tbpA(ap)B(-) > B16B6(Str(r))/tbpA(-)B(ap). Of these transformants, only B16B6(Str(r))/tbpA(ap)B(ap) could grow in the presence of porcine transferrin as the sole iron source, achieving a growth rate similar to that of the B16B6 parent strain in the presence of human transferrin.     

Exploiting protein fluctuations at the active-site gorge of human cholinesterases: further optimization of the design strategy to develop extremely potent inhibitors

Protein conformational fluctuations are critical for biological functions, although the relationship between protein motion and function has yet to be fully explored. By a thorough bioinformatics analysis of cholinesterases (ChEs), we identified specific hot spots, responsible for protein fluctuations and functions, and those active-site residues that play a role in modulating the cooperative network among the key substructures. This drew the optimization of our design strategy to discover potent and reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase (hAChE and hBuChE) that selectively interact with specific protein substructures. Accordingly, two tricyclic moieties differently spaced by functionalized linkers were investigated as molecular yardsticks to probe the finest interactions with specific hot spots in the hChE gorge. A number of SAR trends were identified, and the multisite inhibitors 3a and 3d were found to be the most potent inhibitors of hBuChE and hAChE known to date.     

Traumatic neuroma of the great auricular nerve. A clinical case

A clinical case of neuroma resulting from an amputation which occurred following parotid surgery is reported. The present paper discusses the importance of differential diagnosis relating to this pathology and the surgical techniques that may contribute to setting a limit on its onset. Traumatic neuroma is a possible complication of parotid surgery together with motor deficiencies and Frey's syndrome and the less frequent salivary fistulas. It may also occur following oncological or plastic-reconstructive surgery. During parotidectomy operations, section of the great auricular nerve may be required to facilitate access to the parotid region. This manoeuvre may, however, also be associated with the onset of a neuroma during a time period varying from 2 to 10 years, according to the various authors who have examined the subject. The laterocervical tumefaction associated with neuroma poses a number of diagnostic problems as it has to be differentiated from a likely relapse or possibly from more complex inflammatory or metastatic lymphnodal pathology. A correct clinical and diagnostic multidisciplinary approach is fundamental if the pathology is to be classified in the right way and contributes to alleviating a state of mental unease in the patient that is associated with the sudden appearance of the tumefaction. Sectioning the nervous trunk by means of thermocautery and its subsequent sinking below the main trunk of the sternocleidomastoid muscle, are simple surgical manoeuvres that are useful for preventing the onset of traumatic neuroma.     

T follicular helper (Tfh) cells in normal immune responses and in allergic disorders

Follicular helper T cells (T_fh) are located within germinal centers of lymph nodes. Cognate interaction between T_fh, B cells, and IL‐21 drives B cells to proliferate and differentiate into plasma cells thereby leading to antibody production. T_fh cells and IL‐21 are involved in infectious and autoimmune diseases, immunodeficiencies, vaccination, and cancer. Human peripheral blood CXCR5⁺ CD4⁺ T cells comprise different subsets of T_fh‐like cells. Despite the importance of the IgE response in the pathogenesis of allergic disorders, little is known about the role of follicular and blood T_fh cells and IL‐21 in human and experimental allergic disease. Here, we review recent advances regarding the phenotypic and functional characteristics of both follicular and blood T_fh cells and of the IL‐21/IL‐21R system in the context of allergic disorders.     

Structure-based discovery of a small non-peptidic Neuropilins antagonist exerting in vitro and in vivo anti-tumor activity on breast cancer model

Neuropilin-1/-2 (+33 NRPs), VEGF-A₁₆₅ co-receptors_, are over-expressed during cancer progression. Thus, NRPs targeted drug development is challenged using a multistep in silico/in vitro screening procedure. The first fully non-peptidic VEGF-A₁₆₅/NRPs protein–protein interaction antagonist (IC₅₀ = 34 μM) without effect on pro-angiogenic kinases has been identified (compound-1). This hit showed breast cancer cells anti-proliferative activity (IC₅₀ = 0.60 μM). Compound-1 treated NOG-xenografted mice significantly exerted tumor growth inhibition, which is correlated with Ki-67^low expression and apoptosis. Furthermore, CD31⁺/CD34⁺ vessels are reduced in accordance with HUVEC-tube formation inhibition (IC₅₀ = 0.20 μM). Taking together, compound-1 is the first fully organic inhibitor targeting NRPs.     

Isolated Extrapelvic Endometriosis of the Gluteal Muscle

A 33-year-old woman with a 2-year history of swelling and pain in her buttock and left thigh fluctuating with her menstrual cycle who was becoming progressively disabled was referred to the department of orthopedics. Magnetic resonance imaging (MRI) detected a left buttock lesion of 3 × 2 cm that was initially diagnosed as muscular-fiber laceration with associated hematoma. The worsening of her symptomatology required an ultrasound-guided biopsy of the lesion that revealed endometriosis. Laparoscopy showed the pelvis to be free of gross disease. Hormonal suppression by means of gonadotropin-releasing hormone analog therapy proved adequate in temporarily alleviating symptoms. A year later the patient underwent surgical excision of the buttock lesion, which was effective in alleviating her symptoms for a short period of 10 months. A 1-year follow-up MRI revealed several small endometriotic foci, located among piriformis and obturator internus muscle fibers, which were considered not suitable for surgical removal. The patient is currently on a drug regime for pain management. However, she has experienced permanent muscular damage on her left buttock including significant omolateral gluteus strength reduction, functional impairment (inability to rotate laterally or bend her left leg), and the assumption of an antalgic gait while walking. Because of impairment in her deambulation capability, total physical invalidity was agreed for her by the National Health Care Services.     

ZD1839 (IRESSA), an EGFR-selective tyrosine kinase inhibitor, enhances taxane activity in bcl-2 overexpressing, multidrug-resistant MCF-7 ADR human breast cancer cells

Constitutive bcl-2 overexpression increases the tumorigenic and metastatic potential of doxorubicin-resistant, estrogen-independent, MCF-7 ADR human breast cancer cells. We evaluated the sensitivity to taxanes (paclitaxel, docetaxel and IDN 5109) of 2 bcl-2-overexpressing MCF-7 ADR clones and control neomycin-transfected MCF-7 ADR neo cells. The 2 bcl-2-overexpressing MCF-7 ADR clones were relatively resistant to all 3 taxanes, whereas the MCF-7 ADR neo cells were relatively resistant to paclitaxel and docetaxel, but sensitive to IDN 5109. We found that both MCF-7 ADR neo and bcl-2-overexpressing MCF-7 ADR clones express high levels of the epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor-alpha (TGF-alpha). Therefore, we tested the growth inhibitory effect of ZD1839 (Iressa, AstraZeneca, Macclesfield, UK), an orally active, selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that is in clinical development. ZD1839 inhibited the growth in soft agar of all 3 clones in a dose-dependent manner (IC(50) of approximately 0.1 microm). This effect was accompanied by a dose-dependent inhibition of EGFR tyrosine autophosphorylation and of the production of TGF-alpha, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). To determine whether the blockade of EGFR signaling might affect the sensitivity of bcl-2-overexpressing MCF-7 ADR cells to taxanes, cells were treated with ZD1839 in combination with paclitaxel, docetaxel or IDN 5109, and dose-dependent cooperative growth inhibition as well as apoptosis potentiation were observed. Combined treatment with IDN 5109 and ZD1839 also resulted in a significant inhibition of bcl-2 expression in bcl-2-overexpressing MCF-7 ADR cells. These results demonstrate the ability of ZD1839 to overcome taxane resistance in a model of hormone-independent, multidrug-resistant, human breast cancer.