Suran L. Fernando

A thr357 to ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs atp-induced mycobacterial killing by macrophages

The P2X7 receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X7 receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5′-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X7 polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C → G), which changes Thr357 to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X7 function was measured by ATP-induced ethidium+ influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10–65% in heterozygotes, 1–18% in homozygotes, and 0–10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X7 in either HEK-293 cells or Xenopus oocytes gave P2X7 function of ∼50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X7, restored P2X7 function to near normal in cells heterozygous for T357S and to a value 50–65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X7 function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X7 receptor function.

A Loss-of-Function Polymorphism in the Human P2X7 Receptor Abolishes ATP-Mediated Killing of Mycobacteria

Protective immunity to mycobacterial infections requires activation of the antibacterial mechanisms of infected macrophages. It has previously been reported that ATP treatment of mycobacteria-infected macrophages induces apoptosis mediated via the P2X7 pathway and that this leads to the death of both the host cell and the internalized bacilli. We have recently identified a single nucleotide polymorphism in the P2X7 gene (1513A→C), with 1–2% prevalence in the homozygous state, which codes for a nonfunctional receptor. IFN-γ-primed, mycobacteria-infected macrophages from wild-type individuals were incubated with ATP and this induced apoptosis and reduced mycobacterial viability by 90%. Similar treatment of macrophages from individuals homozygous for the 1513C polymorphism failed to induce apoptosis and did not lead to mycobacterial killing via the P2X7-mediated pathway. These data demonstrate that a single nucleotide polymorphism in the P2X7 gene can allow survival of mycobacteria within infected host cells.

Acute generalised exanthematous pustulosis

Acute generalised exanthematous pustulosis (AGEP) is a severe cutaneous adverse reaction and is caused by drugs in >90% of cases. It is rare, with an incidence of 1–5 patients per million per year. The clinical manifestations are characterised by fever and the rapid appearance of disseminated sterile pustules 3–5 days after the commencement of treatment. It is accompanied by marked neutrophilia. Mucous membranes are not typically involved. The drugs conferring the highest risk of AGEP according to the EuroSCAR study are aminopenicillins, pristinamycin, hydroxychloroquine, antibacterial sulphonamides, terbinafine and diltiazem. The pathogenesis of AGEP involves the initial influx of CD8 cytotoxic T‐cells resulting in the apoptosis of keratinocytes and formation of vesicles. Then CXCL‐8‐producing and granulocyte macrophage‐colony stimulating factor‐producing CD4 cells enter the epidermis, resulting in neutrophil mediated inflammation and the formation of pustules. As a result, the histology reveals intraepidermal, usually subcorneal, pustules and an accompanying neutrophilic and lymphocytic infiltrate. Epicutaneous patch testing may also support the diagnosis by causing a localised pustular reaction 48–96 h after the offending drug is applied. The condition usually resolves by 15 days after the causative drug is withdrawn but oral corticosteroid therapy may be necessary in some individuals. The mortality rate is up to 5% and mostly occurs in elderly people who have significant comorbidities.

Drug-reaction eosinophilia and systemic symptoms and drug-induced hypersensitivity syndrome.

Drug reaction with eosinophilia and systemic symptoms (DRESS), also known as drug‐induced hypersensitivity syndrome (DIHS), is a rare, severe cutaneous adverse reaction characterised by fever, rash, lymphadenopathy, eosinophilia and/or other leukocyte abnormalities, and internal organ involvement and often has a relapsing–remitting course despite withdrawal of the drug. The drugs that are most implicated include aromatic anticonvulsants, allopurinol, sulphonamides, antiretrovirals (abacavir and nevirapine), and minocycline. The pathogenesis of DRESS/DIHS is far from clear but probably involves a combination of impaired pharmacokinetics and the accumulation of drug metabolites, the sequential reactivation of the herpesvirus family and genetic susceptibility conferred by the association with certain human leukocyte antigen (HLA) class I alleles. The strong association between abacavir and HLA‐B*5701 has enabled pharmacogenetics screening to be employed successfully to minimise the occurrence of hypersensitivity. A prolonged course of oral corticosteroids is required to treat DRESS/DIHS, given the relapsing–remitting nature of the condition with i.v. immunoglobulin and valgangciclovir reserved for refractory or life‐threatening cases.

Drug-Induced Hypersensitivity Syndrome With Superficial Granulomatous Dermatitis-A Novel Finding

Drug-induced hypersensitivity syndrome (DIHS) is a rare and potentially fatal reaction characterized by fever, rash, and internal organ involvement that typically occurs between 3 and 6 weeks after commencing the drug. We describe such a case in a 26-year-old woman, who developed fever, exfoliative erythroderma, facial edema, cervical lymphadenopathy, hepatitis, and leukocytosis 6 weeks after commencing carbamazepine for lower back pain. Her serum angiotensin-converting enzyme level was also raised to 144 U/L (8-52 U/L). Skin biopsies demonstrated an unusual superficial dermal perivascular inflammatory infiltrate, which included conspicuous granulomas mixed with moderate numbers of lymphocytes. Eosinophils were not a feature. Her carbamazepine was withdrawn, and oral prednisone was commenced initially at a dose of 1 mg.kg.d and slowly weaned over a 6-week period. Her fever, rash, facial edema, and hepatitis gradually resolved within this period, and her serum angiotensin-converting enzyme level returned to within the normal range. Although the patients clinical course was consistent with a DIHS, it was accompanied by a previously unreported finding of a superficial granulomatous dermatitis. Granuloma formation as a sequel to medication use is a feature of interstitial granulomatous drug reaction. However, interstitial granulomatous drug reaction consists of localized violaceous plaques with a predilection for skin fold areas and liver function abnormalities have not been described. Granulomatous inflammation in other organ systems, including the liver and kidney, has also been described after the use of carbamazepine, but these reactions are not associated with the systemic manifestations observed in DIHS.

Two Case Reports of Life-Threatening Ethanol-Induced Anaphylaxis.

Adverse reactions to alcoholic beverages are common and diverse in aetiology. Ethanol-induced anaphylaxis, however, is a rare but often life-threatening condition that warrants careful evaluation in suspected individuals. We present the cases of two patients who developed urticaria, angioedema and throat constriction within minutes of consuming white wine. Both individuals demonstrated no adverse reaction to double-blind placebo-controlled challenges to metabisulphite or sodium salicylate. However, an open challenge to white wine elicited urticaria in both subjects. This reaction was reproduced with a double-blind placebo-controlled challenge to ethanol and was accompanied by a rise in serum total tryptase levels. Positive skin test responses to 2% acetic acid, a breakdown product of ethanol, were elicited from both patients but not from three normal controls. These two cases demonstrate the need for a systematic approach for the evaluation of allergic reactions to alcohol.

Serum free light chain assay reduces the need for serum and urine immunofixation electrophoresis in the evaluation of monoclonal gammopathy

Background The potential for serum free light chain (sFLC) assay measurements to replace urine electrophoresis (uEPG) and to also diminish the need for serum immunofixation (sIFE) in the screening for monoclonal gammopathy was assessed. A testing algorithm for monoclonal protein was developed based on our data and cost analysis. Methods Data from 890 consecutive sFLC requests were retrospectively analysed. These included 549 samples for serum electrophoresis (sEPG), 447 for sIFE, and 318 for uEPG and urine immunofixation (uIFE). A total of 219 samples had sFLC, sEPG, sIFE and uEPG + uIFE performed. The ability of different test combinations to detect the presence of monoclonal proteins was compared. Results The sFLC κ/λ ratio (FLC ratio) indicated monoclonal light chains in 12% more samples than uEPG + uIFE. The combination of sEPG and FLC ratio detected monoclonal proteins in 49% more samples than the combination of sEPG and sIFE. Furthermore, the sEPG + FLC ratio combination detected monoclonal protein in 6% more samples than were detected by the combined performance of sEPG, sIFE, uEPG and uIFE. However, non-linearity of the assay, the expense of repeat determinations due to the narrow measuring ranges, and frequent antigen excess checks were found to be limitations of the sFLC assay in this study. Conclusion The FLC ratio is a more sensitive method than uIFE in the detection of monoclonal light chains and may substantially reduce the need for onerous 24 h urine collections. Our proposed algorithm for the evaluation of monoclonal gammopathy incorporates the sFLC assay, resulting in a reduction in the performance of labour intensive sIFE and uEPG + uIFE while still increasing the detection of monoclonal proteins.

Validation of a rapid test for HLA-B*58:01/57:01 allele screening to detect individuals at risk for drug-induced hypersensitivity

AIM In prevention of allopurinol and abacavir hypersensitivity, screening HLA-B*58:01/57:01 has been highly recommended prior to commencing these therapies. Therefore, we aimed at developing and validating a rapid and robust screening method for HLA-B*58:01/57:01. MATERIALS & METHODS Real-time polymerase chain reaction with TaqMan probes was employed to detect HLA-B*58:01/57:01. RESULTS The newly developed assay has the sensitivity of 100% (95% CI: 79.4-100.0%), the specificity of 98.8% (95% CI: 93.6-99.9%), the positive predictive value of 94.1% (95% CI: 71.3-99.9%) and the negative predictive value of 100.0% (95% CI: 95.7-100.0%). The lowest limit of detection is 0.04 ng/µl of DNA. CONCLUSION The present method is a rapid and robust assay that is appropriate for screening of HLA-B*58:01/*57:01 alleles.

The utility of specific IgE testing to chlorhexidine in the investigation of perioperative adverse reactions

Anaphylaxis in associationwith anesthesia and surgery is a rare but initial clinical reaction but exhibited a much increased level of potentially life-threatening event. To avoid repeat episodes, it is important to identify the causative agents. Different agents have been implicated as causes of anaphylaxis during anesthesia. The most prominent are the neuromuscular blocking drugs, latex, and antibiotics. Of increasing interest, however, is the antiseptic agent chlorhexidine. Chlorhexidine is an antiseptic found in a wide variety of products in the perioperative setting and in many household products.1,2 Such widespread availability potentially increases the risk that patients might be sensitized before surgery. Skin testing is the standard diagnostic tool used to investigate causative agents in perioperative allergic episodes. In addition to skin testing, laboratory methods for the investigation of chlorhexidine allergy include measurement of allergen specific IgE (sIgE). A commercial assay for sIgE to chlorhexidine is available, but studies evaluating this assay have been limited to date, with only 1 other evaluation in a large patient group.3 Therefore, the aim of this study was to evaluate and optimize chlorhexidine sIgE testing in cases of perioperative allergy. A total of 130 patients were included in this retrospective analysis of routine clinical investigations. All had been referred to the Anaesthetic Allergy Referral Service, Royal North Shore Hospital (Sydney, Australia) for investigation and were selected based on a clinical presentation that included chlorhexidine as a possible causative agent of a perioperative allergic event. Patients were assessed from June 2009 to August 2013 (66 women, 64 men; median age 56.5 years). Intradermal and skin prick testing was performed according to a standardized protocol with all suspected agents to which patients had been exposed (eMethods). Measurement of sIgE to chlorhexidine and total IgE in patient serum was carried out on samples collected at the time of skin testing using the ImmunoCAP system (Phadia AB, Uppsala, Sweden). Statistical analysis was performed using Analyse-it for Excel 2.30 (Microsoft, Redmond, Washington). This included receiver operating characteristic curve analysis and sensitivity and specificity determinations. The study was approved by the local health district human research ethics committee. Of the 130 patients, 19 had positive prick and/or intradermal skin test reactions for chlorhexidine. Negative skin test results were displayed in 111 patients. The median concentration of chlorhexidine sIgE in the patients with positive skin test reactions was 1.23 kUA/L (range 0.14e11.90) compared with the median concentration in the patients with negative skin test reactions of 0.10 kUA/L (range 0.10e3.05). Using the recommended cutoff value of 0.35 kUA/L, sIgE to chlorhexidine showed a sensitivity of 84.2% with reference to skin test results and a specificity of 93.7%. Positive predictive value was 69.6% and negative predictive value was 97.2%. An alternative cutoff threshold of 0.20 kUA/L was determined by receiver operating characteristic analysis. This increased the sensitivity to 94.7% but only slightly decreased the specificity to 90.1%. The corresponding positive predictive value was 62.1% and the negative predictive value was 99.0%. When using only the standard cutoff threshold, 1 patient produced a negative sIgE result on a sample taken at the time of the

Immune mediated neuropathy following checkpoint immunotherapy

Checkpoint immunotherapy has revolutionised cancer therapy and is now standard treatment for many malignancies including metastatic melanoma. Acute inflammatory neuropathies, often labelled as Guillain-Barre syndrome, are an uncommon but potentially severe complication of checkpoint immunotherapy with individual cases described but never characterised as a group. We describe a case of acute sensorimotor and autonomic neuropathy following a single dose of combination ipilimumab and nivolumab for metastatic melanoma. A literature search was performed, identifying 14 other cases of acute neuropathy following checkpoint immunotherapy, with the clinical, electrophysiological and laboratory features summarised. Most cases described an acute sensorimotor neuropathy (92%) with hyporeflexia (92%) that could occur from induction up till many weeks after the final dose of therapy. In contrast to Guillain-Barre syndrome, the cerebrospinal fluid (CSF) analysis often shows a lymphocytic picture (50%) and the electrophysiology showed an axonal pattern (55%). Treatment was variable and often in combination. 11 cases received steroid therapy with only 1 death within this group, whereas of the 4 patients who did not receive steroid therapy there were 3 deaths. In conclusion checkpoint immunotherapy - induced acute neuropathies are distinct from and progress differently to Guillain-Barre syndrome. As with other immunotherapy related adverse events corticosteroid therapy should be initiated in addition to usual therapy.