Patrick J. Tighe

Antimicrobial defensin peptides of the human ocular surface.

BACKGROUND/AIMS The antimicrobial activity of the tear film exceeds the activity of its known constituents. The authors postulate that this excess activity is the result of antimicrobial peptides called defensins, and they aimed to look for defensins in the human eye. METHODS Evidence of defensin production was sought by reverse transcriptase polymerase chain reaction (RT-PCR). Intron spanning primers were designed for β defensins 1 and 2, and α defensins 5 and 6. RT-PCR was performed on cornea, conjunctiva, and lacrimal gland samples, and reaction products were size fractionated and sequenced to confirm their identity. A monoclonal antibody was utilised for the detection of α defensins 1, 2, and 3 in tissue sections and in immunoblots of tears. RESULTS RT-PCR revealed β defensin 1 message in samples of conjunctiva, cornea, and lacrimal gland. β Defensin 2 message was detected in the conjunctiva and cornea but was absent from the lacrimal gland. α Defensin 5 and 6 message was absent in these tissues but α defensins 1, 2, and 3 were detected in normal tears, lacrimal gland, and inflamed conjunctiva by immunochemistry. CONCLUSION The data suggest the human eye innately produces a spectrum of antimicrobial defensin peptides. Defensins hold therapeutic potential in ocular infections as they have a broad spectrum of antimicrobial activity (bacteria fungi and viruses ) and accelerate epithelial healing.

Astrocytes as antigen-presenting cells: expression of IL-12/IL-23

Interleukin‐12 (IL‐12, p70) a heterodimeric cytokine of p40 and p35 subunits, important for Th1‐type immune responses, has been attributed a prominent role in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Recently, the related heterodimeric cytokine, IL‐23, composed of the same p40 subunit as IL‐12 and a unique p19 subunit, was shown to be involved in Th1 responses and EAE. We investigated whether astrocytes and microglia, CNS cells with antigen‐presenting cell (APC) function can present antigen to myelin basic protein (MBP)‐reactive T cells, and whether this presentation is blocked with antibodies against IL‐12/IL‐23p40. Interferon (IFN)‐γ‐treated APC induced proliferation of MBP‐reactive T cells. Anti‐IL‐12/IL‐23p40 antibodies blocked this proliferation. These results support and extend our previous observation that astrocytes and microglia produce IL‐12/IL‐23p40. Moreover, we show that stimulated astrocytes and microglia produce biologically active IL‐12p70. Because IL‐12 and IL‐23 share p40, we wanted to determine whether astrocytes also express IL‐12p35 and IL‐23p19, as microglia were already shown to express them. Astrocytes expressed IL‐12p35 mRNA constitutively, and IL‐23 p19 after stimulation. Thus, astrocytes, under inflammatory conditions, express all subunits of IL‐12/IL‐23. Their ability to present antigen to encephalitogenic T cells can be blocked by neutralizing anti‐IL‐12/IL‐23p40 antibodies.

ELISA in the multiplex era: Potentials and pitfalls

Multiplex immunoassays confer several advantages over widely adopted singleplex immunoassays including increased efficiency at a reduced expense, greater output per sample volume ratios and higher throughput predicating more resolute, detailed diagnostics and facilitating personalised medicine. Nonetheless, to date, relatively few protein multiplex immunoassays have been validated for in vitro diagnostics in clinical/point‐of‐care settings. This review article will outline the challenges, which must be ameliorated prior to the widespread integration of multiplex immunoassays in clinical settings: (i) biomarker validation; (ii) standardisation of immunoassay design and quality control (calibration and quantification); (iii) availability, stability, specificity and cross‐reactivity of reagents; (iv) assay automation and the use of validated algorithms for transformation of raw data into diagnostic results. A compendium of multiplex immunoassays applicable to in vitro diagnostics and a summary of the diagnostic products currently available commercially are included, along with an analysis of the relative states of development for each format (namely planar slide based, suspension and planar/microtitre plate based) with respect to the aforementioned issues.

Treatment of Erdheim-Chester disease with cladribine: a rational approach

Erdheim-Chester disease is a rare, life threatening lipoid granulomatosis1 with fewer than 100 cases described in the world literature. The disease typically affects the long bones and symmetrical sclerosis of the diaphyseal and metaphyseal regions is pathognomonic. Extraskeletal manifestations may affect the lungs, pericardium, aorta, retroperitoneum, skin, and orbits and diabetes insipidus occurs in approximately 30% of cases. Erdheim-Chester disease is characterised microscopically by an infiltrate of lipid laden foamy macrophages (histiocytes), scattered Touton giant cells, chronic inflammatory cells, and fibrosis. The foamy macrophages can be distinguished from Langerhans cells on the basis of negative results on staining for S-100 protein and CD1a. Treatment of the disease has been on an ad hoc basis and no treatment regimen has been shown to be clearly superior. This study documents the clinical findings in a patient with Erdheim–Chester disease, investigates the pathogenesis, and provides a rational basis for effective treatment. This white man, aged 45, developed aching in his legs, night sweats, lethargy, and impotence in October 1988, for which no cause was found. His night sweats resolved by July 1989 and he was discharged. He presented in November 1990 with reduced vision (6/9) in the left eye, bilateral proptosis of 12 months’ duration, chemosis, ophthalmoplegia, and optic disc oedema. He still had sexual dysfunction and lethargy and now also had leg oedema and thrombocythaemia. At that time his thyroid function was normal, but erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) were moderately elevated. A computed tomography (CT) scan of the orbits showed bilaterally enhancing masses lying predominantly within the muscle cone and encasing both optic nerves. An orbital biopsy in November 1990 showed an inflammatory picture. There was no evidence of vasculitis on muscle biopsy and a clinical diagnosis of orbital pseudotumour was made. He was initially treated …

Mutant forms of tumour necrosis factor receptor I that occur in TNF‐receptor‐associated periodic syndrome retain signalling functions but show abnormal behaviour

Tumour necrosis factor (TNF)‐receptor‐associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder involving autosomal‐dominant missense mutations in TNF receptor superfamily 1A (TNFRSF1A) ectodomains. To elucidate the molecular effects of TRAPS‐related mutations, we transfected HEK‐293 cells to produce lines stably expressing high levels of either wild‐type (WT) or single mutant recombinant forms of TNFRSF1A. Mutants with single amino acid substitutions in the first cysteine‐rich domain (CRD1) were produced both as full‐length receptor proteins and as truncated forms lacking the cytoplasmic signalling domain (Δsig). High‐level expression of either WT or mutant full‐length TNFRSF1A spontaneously induced apoptosis and interleukin‐8 production, indicating that the mutations in CRD1 did not abrogate signalling. Consistent with this, WT and mutant full‐length TNFRSF1A formed cytoplasmic aggregates that co‐localized with ubiquitin and chaperones, and with the signal transducer TRADD, but not with the inhibitor, silencer of death domain (SODD). Furthermore, as expected, WT and mutant Δsig forms of TNFRSF1A did not induce apoptosis or interleukin‐8 production. However, whereas the WT full‐length TNFRSF1A was expressed both in the cytoplasm and on the cell surface, the mutant receptors showed strong cytoplasmic expression but reduced cell‐surface expression. The WT and mutant Δsig forms of TNFRSF1A were all expressed at the cell surface, but a proportion of the mutant receptors were also retained in the cytoplasm and co‐localized with BiP. Furthermore, the mutant forms of surface‐expressed Δsig TNFRSF1A were defective in binding TNF‐α. We conclude that TRAPS‐related CRD1 mutants of TNFRSF1A possess signalling properties associated with the cytoplasmic death domain, but other behavioural features of the mutant receptors are abnormal, including intracellular trafficking and TNF binding.

Quantitative analysis of IL‐10 and IFN‐γ mRNA levels in normal cervix and human papillomavirus type 16 associated cervical precancer

Human papillomavirus type 16 is a major factor in cervical carcinogenesis. Inappropriate cytokine synthesis may direct the local immune response away from a type‐1 (cellular) pattern and may subsequently contribute to the development and progression of precancer. Quantitative reverse transcription–polymerase chain reaction (RT‐PCR) using a competitive mimic was carried out to determine type‐1 (interferon gamma (IFN‐γ)) and type‐2 (interleukin‐10 (IL‐10)) cytokine mRNA levels in whole cervical specimens (without microdissection) from seven normal and nine HPV‐16 positive CIN formalin‐fixed paraffin‐embedded tissues. Microdissection was used to measure separately the epithelial and sub‐epithelial levels of IFN‐γ and IL‐10 mRNAs in 11 specimens of normal cervix and 25 HPV‐16 positive CIN (nine CIN 1, seven CIN 2 and nine CIN 3). IFN‐γ mRNA was lower in CIN than normal (p=0.04). IL‐10 mRNA level in CIN was significantly higher (p=0.005) than in normal cervix (before microdissection). Epithelial IFN‐γ mRNA showed a significant decrease in all grades of CIN (median=3.58) compared with normal (7.74) (p<0.05), but there was no significant difference between the grades. A significant decrease in sub‐epithelial IFN‐γ mRNA was found in CIN 1(9.81), CIN 2 (3.82) and CIN 3 (4.62) compared with normal cervix (27.35) (p<0.05). Also, sub‐epithelial IFN‐γ mRNA was significantly lower in CIN 2 and CIN 3 than in CIN 1 (p=0.005 and 0.0005, respectively). IL‐10 was detected in the epithelium of only one of 11 normal and one of 25 CIN, but sub‐epithelial IL‐10 was significantly higher in CIN 2 (0.08) and CIN 3 (0.26) than in normal (0.00) (p=0.036 and 0.0032, respectively). There was no significant difference in the sub‐epithelial level of IL‐10 between normal and CIN 1 (0.00) (p=0.96). Our results suggest that reduced epithelial and sub‐epithelial IFN‐γ, as well as increased sub‐epithelial IL‐10 synthesis may play a role in the development and progression of HPV‐16 associated cervical precancer. Copyright

Role of interleukin-6 in a patient with tumor necrosis factor receptor-associated periodic syndrome: assessment of outcomes following treatment with the anti-interleukin-6 receptor monoclonal antibody tocilizumab.

In this report, we describe treatment outcomes in the first case of a patient with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) treated with the anti-interleukin-6 (anti-IL-6) receptor monoclonal antibody tocilizumab. Since IL-6 levels are elevated in TRAPS, we hypothesized that tocilizumab might be effective. The patient, a 52-year-old man with lifelong TRAPS in whom treatment with etanercept and anakinra had failed, was administered tocilizumab for 6 months, and the therapeutic response was assessed by measurement of monocyte CD16 expression and cytokine levels. Following treatment, the evolving acute attack was aborted and further attacks of TRAPS were prevented. The patient did not require corticosteroids and showed significant clinical improvement in scores for pain, stiffness, and well-being. Moreover, the acute-phase response diminished significantly with treatment. Monocyte CD16 expression was reduced and the numbers of circulating CD14+CD16+ and CD14++CD16- monocytes were transiently decreased. However, cytokine levels were not reduced. This case supports the notion of a prominent role for IL-6 in mediating the inflammatory attacks in TRAPS, but blockade of IL-6 did not affect the underlying pathogenesis. These preliminary findings require confirmation.

Reinfection with hookworm after chemotherapy in Papua New Guinea

Reinfection with hookworm (Necator americanus) following chemotherapy was studied over 2 years in a rural village in Madang Province, Papua New Guinea. The prevalence of hookworm infection had returned to pre-treatment levels after 2 years, and the geometric mean hookworm burden had returned to 58% of the pre-treatment value. The rate of acquisition of adult worms was independent of host age, and was estimated as a geometric mean of 2.9-3.3 worms/host/year (arithmetic mean 7.9-8.9 worms/host/year). There was significant predisposition to hookworm infection; the strength of this predisposition did not vary significantly between age or sex classes.

In situ gelling hydrogels incorporating microparticles as drug delivery carriers for regenerative medicine

Aqueous solutions of blends of biodegradable triblock copolymers, composed of poly(D,L-lactide-co-glycolide) (PLGA) and poly(ethylene glycol) (PEG) with varied D,L-lactide to glycolide ratios, displayed thermosensitivity and formed a gel at body temperature. The gel window of the blend solutions could be tuned by varying the blending ratio between the two components. Furthermore, the storage modulus of the resultant hydrogel from the copolymer blends at body temperature was higher than that of each individual component. Incorporation of poly(D,L-lactide) (PDLLA) microparticles (0.5-40% w/v) within the in situ gelling hydrogel did not change the sol-gel transition temperatures of the polymer solutions, while the mechanical strength of the resultant hydrogels was enhanced when the content of the microparticles was increased up to 30% and 40%. Incorporation of proteins into both the gel and microparticle components resulted in composites that controlled the kinetics of protein release. Protein within the gel phase was released over a 10-day period whilst protein in the microparticles was released over a period of months. This system can be used to deliver two drugs with differing release kinetics and could be used to orchestrate tissue regeneration responses over differing timescales.

Differential gene expression during meningeal-meningococcal interaction: evidence for self-defense and early release of cytokines and chemokines.

ABSTRACT Using microarray technology, we studied the early differential expression of 3,528 genes in human meningothelial cells in response to meningococcal challenge. Thirty-two genes were up-regulated, and four were down-regulated. Those up-regulated included the tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8 (but not IL-1β) genes, suggesting that meningeal cells may be a local and early source of these cytokines. Also, a trend in up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes was observed. This is the first evidence that meningothelial cells may mount cytoprotective responses to pathogenic bacteria.