Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis.

TM4 is a lytic mycobacteriophage which infects mycobacteria of clinical importance. A luciferase reporter phage, phAE40, has been constructed from TM4 and was previously shown to be useful for the rapid detection and drug susceptibility testing of Mycobacterium tuberculosis. However, the lytic nature of the phage results in a loss of detectable light output and limits the sensitivity of detection. We describe several strategies aimed at improving the luciferase activity generated by TM4 luciferase phages, including (i) varying the position of the luciferase gene in the phage genome, (ii) isolating host-range mutants of the phage, and (iii) introducing temperature-sensitive mutations in the phage such that it will not replicate at the infecting temperature. Several new phages generated by these methods show increased intensity of luciferase production compared to the first-generation reporter phage phAE40, and one phage, phAE88, also demonstrates an enhanced duration of luciferase activity. This has allowed the detection of as few as 120 BCG cells and the determination of drug susceptibilities of M. tuberculosis in as little as 1 day.     

Oligoclonal populations of T and B cells in a case of angioimmunoblastic T-cell lymphoma predominantly infiltrated by T cells of the VB5.1 family.

AIMS: Immunohistological and molecular characterisation of a case of peripheral T-cell lymphoma (PTCL) of angioimmunoblastic T-cell lymphoma (AILD) type. METHODS: Frozen and paraffin wax sections of the diagnostic lymph node were stained with a panel of T- and B-cell lineage monoclonal antibodies. DNA was isolated from the paraffin wax embedded biopsy material for T-cell receptor (TCR) and immunoglobulin (Ig) PCR amplification, and resultant PCR products were cloned and sequenced. RESULTS: Immunohistological analysis of the presenting lymph node was consistent with an extensive infiltrate of pleomorphic CD3+CD8+ lymphocytes. Most (>80%) of these infiltrating CD3+ cells were also positive for the TCR VB5.1 gene family product, and were shown to be oligoclonal by TCRB PCR amplification and sequencing. Three oligoclones of B cells were also demonstrable by PCR amplification with Ig heavy chain primers and sequencing, a finding at variance with the diagnosis of AILD. CONCLUSIONS: These data demonstrate the complexity and heterogeneity of PTCL which require extensive histological examination and molecular characterisation.     

Recurrent dislocation of the patella and the Goldthwait operation

In this study 17 patients with recurrent dislocation of the patella were followed up 10 years after their Goldthwait operation. The subjective and clinical findings were excellent or good in 70%. X-radiographs indicated osteoarthritis of the femoropatellar joint in 60%. Concerning the aetiopathological factors, we found an increased external torsion of the afflicted extremity (measured by computed tomography).     

Adverse reactions following pulsed tunable dye laser treatmentof port wine stains in 701 patients

The pulsed tunable dye laser (PTDL) is generally considered to have a very low incidence of adverse effects, allowing it to become the treatment of choice for the majority of port wine stains (PWS). The low incidence of adverse effects has led to difficulties in determining the true incidence and type of adverse effect seen with this laser. We therefore undertook a retrospective study of 701 patients with PWS, who received 3877 full treatments to determine the incidence and type of adverse effects seen following treatment with the PTDL. Blistering and crusting were seen in 5·9% and 0·7% of patients, respectively, but were transient events which usually healed without permanent sequelae. Hyperpigmentation was the most frequently observed adverse effect seen in 9·1% of patients but generally showed gradual resolution over 6–12 months. Hypopigmentation was infrequent, seen in 1.4% of patients. The most significant adverse effects were atrophic and hypertrophic scarring seen in 4·3% and 0·7% of patients, respectively. Our observations show that there is a small but definite risk of atrophic scarring with a predisposition for younger patients. Hypertrophic scarring can occur albeit rarely and there may be a predisposition towards the neck. In most cases test areas were not predictive of scarring. This underlines the need for a full discussion of scarring risk in patients with PWS undergoing treatment with the PTDL.     

Bullous pemphigoid in association with cutaneous lesions specific to a myelodysplastic syndrome

Specific cutaneous lesions are a rare occurrence in myelodysplastic syndromes (MDS). The concurrent association of blistering skin lesions similar to those in bullous pemphigoid (BP), even though a rare event, suggests that BP may be a paraneoplastic syndrome. We report an 86-year-old man who had a refractory anaemia with excess bone marrow blasts in transformation, who developed a generalized pruritic blistering eruption. Immunohistopathological tests showed subepidermal blisters with linear deposits of IgG and C3 along the basement membrane zone of the epidermis surrounding a tumoral dermal infiltrate of CD13+ and CD15+ cells. Immunoblotting studies using epidermal extracts revealed circulating IgG antibodies against three protein bands: a 210–215 kDa band. a 180kDa band which co-migrated with the BP 180 antigen, and a 190kDa band. The tumour infiltrate may have revealed antigenic determinants which led to the onset of BP. The concept of paraneoplastic pemphigoid remains to be either confirmed or invalidated by further epidemiological studies.     

Effect of UVB 311 nm irradiation on normal human skin

Ultraviolet radiation B (UVB) on the skin induces erythema, inflammation and modifications of the immune system. These changes have been reported after excessive short-term or long-term exposure to broad spectrum UVB. In this study, we examined the effects of local repetitive UVB irradiation of 311 nm wavelength on the skin of seven young volunteers. Skin biopsies were taken before and after UVB irradiation, and we immunohistochemically analyzed the expression of CD1a and HLA-DR antigens of Langerhans cells (LC), the possible infiltration of dermis/epidermis by CD11b macrophages, the modifications or the induction of intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1) involved in the binding of leukocytes to the endothelial surface and the development of perivascular infiltrates of LFA-1⁺ mononuclear cells. We also determined the expression of substance P receptors (SPR) using biotinylated substance P (SPB). Exposure of UVB 311 nm induced a drastic reduction of CD1a⁺ cells and a moderate increase of HLA-DR⁺ dendritic cells in the epidermis without infiltration by CD11b macrophages. An increase of the binding of SPB to upper layer epidermal cells was noted in five of seven biopsies. In the dermis, vessel-associated ICAM-1 expression increased and an induction of E-selectin occurred on nearly 20 to 40% of endothelial cells, but VCAM-1 expression remained undetectable. The percentage of LFA-1⁺ cells did not change significantly after irradiation. These observations may be compatible with a selective role of UVB 311 nm on the skin immune response.     

Effects of capsaicin in temporomandibular joint arthritis in rats

Temporomandibular joint (TMJ) arthritis was induced in female Lewis rats by unilateral injection of a suspension of heat-killed Mycobacterium butyricum in paraffin oil into the TMJ. Control rats received paraffin oil by the same route. Arthritic and control rats were pretreated either with capsaicin or denervation of the mandibular branch of the trigeminal nerve. Tissues were collected for neuropeptide extraction and analysed by radioimmunoassay and reverse-phase high-performance liquid chromatography. In all groups, the levels of substance P- (SP), calcitonin gene-related peptide- (CGRP) and neuropeptide Y- (NPY) like immunoreactivity (LI) were higher in the trigeminal ganglia than in the TMJs. In control rats, capsaicin significantly lowered the levels of SP-LI in the trigeminal ganglia and TMJ, but not CGRP-LI and NPY-LI. In the arthritic rats, capsaicin pretreatment significantly lowered the SP-LI and CGRP-LI in the trigeminal ganglia and TMJ, but not the NPY-LI. In the trigeminal ganglia the unilateral denervation significantly lowered SP-LI in control rats, and in arthritic rats SP-LI and CGRP-LI. On the denervated side of the arthritic TMJ, NPY-LI, SP-LI and CGRP- LI were significantly lowered as compared to the arthritic control rats and to the contralateral side. In this rat model, pretreatment with capsaicin and surgical denervation decreased the neuropeptide content in the trigeminal ganglia and the TMJ. The results clearly demonstrate a close interaction between increased neuropeptide release from sensory and sympathetic neurones after induction of arthritis in the rat.     

Periapical radiographic techniques during endodontic diagnosis and treatment

The objective of this article is to describe various radiographic projections which can be used during endodontic therapy. Changes to the angulation of the X-ray beam in relation to the teeth and film can help diagnosis and treatment by producing images which provide additional information not always visible on radiographs taken with standard angulations. For example, changes in angulation can be useful to determine the number and curvature of roots and canals, to identify superimposed roots and to distinguish between anatomical landmarks and apical pathology. Although use of such techniques increases the diagnostic yield of films, it must be appreciated that such views lead to images that are less distinct because of inherent image distortion. Nevertheless, use of the various techniques during endodontics can provide substantial benefit for clinicians in their daily practice.