Covalent penicillin-protein conjugates elicit anti-drug antibodies that are clonally and functionally restricted.

Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although ß-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical ß-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)-regardless of CDRH3 length-limits cross-reactivity against other ß-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the 'germline-guided reverse engineering' of such drugs away from unwanted immune responses.

The anti-penicillin antibodies levels in sensitive and insensitive people to intradermal skin test.

BACKGROUND: The hypersensitivity reaction to penicillin is a public health problem. Immunological responses to penicillin and other beta-lactam antibiotics can be classified into immediate and non-immediate responses. The immediate hypersensitivity is mediated by IgE; however, the non-immediate sensitivity is facilitated by other isotypes of antibody or T lymphocytes. OBJECTIVE: This research detected the non-IgE antibody value against penicillin in allergic and normal people. METHODS: Thirty-eight samples from patients with positive or negative intradermal skin testing results of penicillin allergy were included in this study. The total antibody and IgM levels against penicillin G were defined by in-house ELISA test. RESULTS: The results showed a significant (P< 0.05) elevation in total immunoglobulin and non-IgM anti-penicillin antibody of sensitive groups; however, the anti-penicillin IgM was significantly greater in non-sensitive peoples. CONCLUSIONS: Although the sensitized people to penicillin cannot be certainly detected with the total antibody, specific IgG and IgM value against penicillin, these values are good indicators for prediction of immediate and late response of the immune system to penicillin.

Identification of T-cell epitopes from benzylpenicillin conjugated to human serum albumin and implication in penicillin allergy.

BACKGROUND: There is in vitro evidence that T cells from allergic patients react to benzylpenicillin-human serum albumin (BP-HSA) bioconjugates. Our group has recently shown the existence of naïve CD4+ T cells recognizing BP-HSA in healthy donors. However, BP-haptenated peptides from HSA participating in the immunization of allergic patients have never been identified. The purpose of the present study is to identify immunodominant BP-haptenated peptides from HSA involved in immunization of patients to BP and to refine the frequency calculation of naïve CD4+ T cells recognizing BP. METHODS: Co-cultures were established with CD4+ T cells from non-allergic donors and mature autologous dendritic cells (DCs) loaded with BP-HSA or BP-haptenated peptides from HSA. The CD4+ T-cell response specific for BP-HSA or for individual BP-haptenated peptides was measured using an interferon-γ (IFN-γ) ELISpot assay. The frequency of BP-specific CD4+ T cells was then calculated using the Poisson distribution. BP-HSA and BP-haptenated peptides recognition by allergic patients was evaluated on peripheral blood mononuclear cells (PBMCs) using a lymphocyte transformation test (LTT). RESULTS: Results showed that BP-HSA and BP-haptenated peptides were recognized by naïve T cells from 15/16 and 13/14 tested healthy donors, respectively. Most donors responded to 3 peptides with BP covalently bound on lysines 159, 212, and 525. Two of these benzylpenicilloylated peptides (lysines 159 and 525) were also found to induce PBMCs proliferation in patients with allergic reaction to penicillins. CONCLUSION: This study identifies and characterizes for the first time the BP-haptenated peptides from HSA involved in the immunization of patients to penicillins.

Decline in 2 Serial Postmortem Tryptase Measurements Beyond 72 Hours After Death in an Antibiotic-Related Anaphylactic Death.

Anaphylaxis can be difficult to diagnose in the postmortem setting. Postmortem tryptase is a widely used ancillary test in aiding the diagnosis in which an elevation supports a death from anaphylaxis. Postmortem tryptase can be difficult to interpret, and the effects of postmortem kinetics are not fully understood. Clinically, mast cell tryptase returns to baseline 24 to 72 hours after an anaphylactic stimulus. We report another anaphylactic death from antibiotic administration in which 2 serial postmortem total tryptase measurements at 3 days (72 hours) and 6 days (144 hours) after death declined from 522 µg/L to 300 µg/L (baseline, 5.6 µg/L). The declination appears to be slower than what is expected in the clinical setting. This case highlights yet another example of the difficult and complex interaction of postmortem interval on postmortem tryptase, especially in an anaphylactic death. We suggest that early blood sampling and serial tests be performed if possible in suspected anaphylactic death.

Life-Threatening Atypical Case of Acute Generalized Exanthematous Pustulosis.

Antibiotics are known to cause severe cutaneous adverse reactions, such as the rare acute generalized exanthematous pustulosis (AGEP). Unlike Stevens-Johnson syndrome or toxic epidermal necrolysis, AGEP is rarely life-threatening. Systemic involvement is not typical, and if present usually coincides with a mild elevation of the hepatic enzymes and a decrease in renal function. Hence, AGEP is known to have a good prognosis and to be life-threatening only in elderly patients or patients with chronic diseases. Herein, we report a case of AGEP in a young healthy male leading to systemic inflammatory response syndrome and to treatment in an intensive care unit after being treated with 5 different antibiotics. Initial symptoms were not indicative for AGEP and the patient's course of disease led promptly to critical cardiorespiratory symptoms and systemic inflammatory response syndrome. We assume that the administration of the 5 different antibiotics resulted in type IV allergy as well as secondary infection with Enterococcus faecium and Staphylococcus aureus, while the underlying periodontitis also contributed to the severity of this case.

A Rapid and Sensitive Assay for the Detection of Benzylpenicillin (PenG) in Milk.

Antibiotics, such as benzyl-penicillin (PenG) and cephalosporin, are the most common compounds used in animal therapy. Their massive and illegal use in animal therapy and prophylaxis inevitably causes the presence of traces in foods of animal origin (milk and meat), which creates several problems for human health. With the aim to prevent the negative impact of ß-lactam and, in particular, PenG residues present in the milk on customer health, many countries have established maximum residue limits (MRLs). To cope with this problem here, we propose an effective alternative, compared to the analytical methods actually employed, to quantify the presence of penicillin G using the surface plasmon resonance (SPR) method. In particular, the PenG molecule was conjugated to a protein carrier to immunize a rabbit and produce polyclonal antibodies (anti-PenG). The produced antibodies were used as molecular recognition elements for the design of a competitive immune-assay for the detection of PenG by SPR experiments. The detection limit of the developed assay was found to be 8.0 pM, a value much lower than the MRL of the EU regulation limit that is fixed at 12 nM. Thus, our results clearly show that this system could be successfully suitable for the accurate and easy determination of PenG.

Immunosensor for trace penicillin G detection in milk based on supported bilayer lipid membrane modified with gold nanoparticles.

In this work, we developed an immunosensor for electrochemical detection of penicillin G at trace level. The biosensor was fabricated by immobilizing anti-penicillin G in a supported bilayer lipid membrane (s-BLM) modified with gold nanoparticles, and the modified electrodes were characterized by the scanning electron microscope (SEM), cyclic voltammetry and electrochemical impedance spectroscopy. The biosensor was able to detect penicillin G with a linear correlation ranging from 3.34×10(-3)ng/L to 3.34×10(3)ng/L and a detection limit of 2.7×10(-4)ng/L, much lower than the maximum residue limit (MRL) of penicillin G in milk (4ppb, equal to 4×10(3)ng/L) set out by the European Union. The mean coefficient variation (CV) of the intra-assays and the inter-assays were 5.4% and 7.7%, respectively. In addition, the concentration of penicillin G in milk samples determined by this biosensor was in good agreement with that determined by high performance liquid chromatography (HPLC) assay.

Selective immediate responders to amoxicillin and clavulanic acid tolerate penicillin derivative administration after confirming the diagnosis.

BACKGROUND: An increasing number of patients show immediate selective hypersensitivity reactions to clavulanic acid (CLV) and amoxicillin (AX), probably due to their increased prescription. The maintenance of this response should be established. OBJECTIVE: To assess that the immediate hypersensitivity selective response to AX or to CLV is maintained after repeated administration of penicillin G (PG)/penicillin V (PV) and AX. METHODS: Patients with proven immediate hypersensitivity to AX (Group A) or CLV (Group B) were included. Diagnosis was performed using skin tests with major and minor determinants of PG (PPL/MDM), AX and CLV and by drug provocation test (DPT) if required. Selectivity was established by confirming tolerance to PG/PV (Group A) and to PG/PV and AX (Group B). The maintenance of the selective response was verified by repeating DPT, 15 days after the initial investigation, with the same procedure. RESULTS: Of 51 patients, 78% belonged to Group A and 22% to Group B. Most had anaphylaxis. In Group A, 72% were skin test positive; 28% required DPT. In Group B, 63% were skin test positive; 37% required DPT. Only two AX-selective cases developed positive responses after re-provocation with PG/PV. No cases selective for CLV developed a positive response to PG, PV or AX. DISCUSSION: The selective response to AX appears consistent, and a response to penicillin determinants only develops in a minority of cases. For the case of CLV, the selective response appears not to be modified by exposure to penicillin determinants, meaning that patients with CLV allergy can take penicillin derivatives safely.

Recognition of multiepitope dendrimeric antigens by human immunoglobulin E.

In vitro drug allergy tests have limited sensitivity, partly due to a poor understanding of the immunological recognition of in vitro drug-protein conjugates. We have designed and synthesized multivalent mono- and bi-epitope dendrimeric antigen (DeAn) conjugates and studied their chemical and tridimensional structures. We describe differences in the spatial distribution and conformation of these conjugated epitopes for the first time: a partially hidden benzylpenicilloyl and a more exposed amoxicilloyl. Our data suggest that DeAn conjugates provide a useful model for studying IgE recognition in patients who suffer from an allergic reaction to benzylpenicillin and/or amoxicillin. 1D and 2D NMR, MDS and immunochemical studies provide evidence that both antigen composition and tridimensional distribution play key roles in IgE-antigen recognition. Bi-epitope DeAn conjugates could potentially allow the diagnosis of patients allergic to any of these two drugs with a single test and represent the basis for a broadly-applicable in vitro assay. From the clinical editor: The prevalence of allergic drug reactions is rising and there is an imperative need to identify patients at risk. In this interesting and important article, the authors developed a novel method for detecting drug specific IgE antibodies, responsible for allergic reactions, by using multivalent mono- and bi-epitope Dendrimeric Antigen (DeAn) conjugates. The continued success of this research may pave way of eventual development of a simple diagnostic test.

Skin testing only with penicillin G in children with a history of penicillin allergy.

BACKGROUND: The absence of commercially available penicilloyl-polylysine (PPL) for most of the last decade severely hampered the practice of penicillin allergy evaluation because skin testing without PPL is reported to have a poor negative predictive value (NPV). OBJECTIVE: To determine the safety and NPV of skin testing without PPL using only penicillin G followed by a 3-dose graded challenge to the incriminated penicillin in children with a history of penicillin allergy. METHODS: Patients evaluated for a history of penicillin allergy at the CHU Sainte-Justine Allergy Clinic between December 2006 and December 2009 were skin tested only with penicillin G and underwent a 3-dose graded challenge to the culprit penicillin if the skin test result was negative. RESULTS: Among 563 patients skin tested to penicillin G, 185 (33%) had a positive skin test result. These patients had a shorter interval between the initial reaction and skin testing compared with patients with a negative skin test result (P = .03). A total of 375 of 378 patients (99%) with a negative skin test result were challenged and 18 (4.8%) reacted, translating into a NPV of 95.2% (95% confidence interval [CI], 92.5%-97.1%). Three of 17 patients with a history of anaphylaxis and a negative skin test result reacted to challenge (NPV, 82.4%; 95% CI, 59.0-93.8%). All challenge reactions were mild and resolved promptly with treatment. CONCLUSION: Among children with a history of penicillin allergy, skin testing only with penicillin G followed by a 3-dose graded challenge to the incriminated penicillin is safe and yields a good NPV. This approach could be useful when PPL is unavailable.

IgE to penicillins with different specificities can be identified by a multiepitope macromolecule: Bihaptenic penicillin structures and IgE specificities.

Quantitation of specific IgE by immunoassay is a recommended in vitro test for the diagnosis of immediate hypersensitivity reactions to betalactams (BLs), particularly when skin test results are negative. IgE antibodies that recognize the common nuclear structure of all BLs or the specific side chain structure can be mainly distinguished by immunoassays. The aim of this study was to develop an immunoassay system to detect IgE antibodies with different specificities. Cellulose discs conjugated with benzylpenicillin (BP), amoxicillin (AX) or both drugs, with poly-l-lysine (PLL) as carrier molecule, were used as solid phases in the radioallergosorbent test (RAST). Direct and inhibition radioimmunoassay studies were made to verify the structures recognized by serum IgE antibodies from penicillin-allergic patients. Our results indicated that the addition of both haptens did not decrease the capacity to capture IgE when serum specific to either BP or AX was used, at least in terms of sensitivity. In addition, the inclusion of two haptens improved significantly the levels of IgE detection in patients who recognized both BP and AX. Therefore, the use of a solid phase with a carrier molecule conjugated with two determinants (AX and BP) is helpful to recognize IgE antibodies against either of these determinants and is useful for screening sera with different specificities.

Identification and frequency of circulating CD4(+) T lymphocytes specific to Benzylpenicillin in healthy donors.

BACKGROUND: Drug hypersensitivity is known to rely on a drug-specific T-cell response. Amplitude of antigen-specific T-cell response is partly controlled by the size of the antigen-specific naïve CD4(+) T-cell repertoire, but estimate of this repertoire has never been investigated for allergenic drugs. The purpose of this study was to evaluate the frequency of benzylpenicillin-specific CD4(+) T lymphocytes in healthy donors. METHODS: Co-cultures were established with CD4(+) T lymphocytes from healthy donors and mature autologous dendritic cells loaded with benzylpenicillin coupled to human serum albumin. CD4(+) T lymphocytes were stimulated once a week for 4 weeks with benzylpenicillin coupled to human serum albumin. The CD4(+) T-cell response was measured using an interferon-γ ELISPOT assay. Frequency of benzylpenicillin-specific naive CD4(+) T lymphocytes was then calculated using the Poisson distribution law. RESULTS: Results showed the presence of benzylpenicillin-specific CD4(+) T lymphocytes in 9 of 10 tested healthy donors irrespective of their HLA typing, with a mean frequency of 0.29 cells per million of CD4(+) T cells. Experiments performed on naive (CD45RA(+) ) and on memory (CD45RO(+) ) CD4(+) T lymphocytes showed that these benzylpenicillin-specific CD4(+) T lymphocytes belonged to the naive T-cell subpopulation. CONCLUSION: This study showed for the first time the existence of naive CD4(+) T lymphocytes specific to benzylpenicillin in healthy donors.

Non-antibiotic properties of tetracyclines as anti-allergy and asthma drugs.

All available therapies for human allergic disease target IgE mediated pathologic responses after IgE has been produced. We are developing tetracyclines as anti-allergy drugs to prevent IgE production, based on our findings that minocycline or doxycycline treatment of allergic asthmatic humans significantly improves their asthma symptoms, reduces their oral steroid requirements, and strongly suppresses their ongoing IgE responses (ELISA, mast cell mediated cutaneous late phase responses); the tetracyclines also strongly suppress peak IgE responses of BPO-KLH sensitized mice (ELISPOT assay, ELISA, skin tests). The antibiotic activity of the tetracyclines is not required for suppression of IgE responses; inclusion of minocycline or doxycycline in sterile culture prevents anti-CD40/IL-4 mediated induction of memory IgE responses by PBMC of allergic asthmatic patients (ELISA), and induction of specific memory IgE responses by spleen cells of BPO-KLH sensitized mice (ELISPOT assay, ELISA). The tetracyclines affect an epsilon specific pathway because IgM, IgG and IgA responses did not decrease. Further, in humans, DTH responses to recall antigens did not decrease. In related studies, we found that two distinct T cell subsets: CD4+CD60 negative and CD8+CD60+ (CD60 is a ganglioside) (humans) and CD4+ Asialo GM1 ganglioside negative and CD8+Asialo GM1 ganglioside+ (mice), both are required for induction of memory IgE responses. Phosphorylated (phos) p38 MAP kinase, but not phos ERK or phos JNK expression by CD4+ and CD8+, including CD8+CD60+, T cells is increased in allergic asthmatic humans, as is IL-4 and IL-10 production. The tetracyclines appear to target T cell pathways to induce suppression of IgE responses because they suppress phos p38 MAP kinase expression by both CD4+ and CD8+, including CD8+CD60+, T cell subsets, and IL-4 and IL-10, while upregulating IL-2 and IFN gamma, and suppressing IgE responses. Our finding that tetracyclines do not require antibiotic activity to suppress IgE responses opens the door to development of new tetracycline-based and other therapeutics for human allergic disease.

Hypersensitivity reactions to penicillins: studies in a group of patients with negative benzylpenicillin G skin test.

BACKGROUND: Although skin tests are usually employed to evaluate current penicillin allergy status, a negative result does not exclude hypersensitivity. There is a need for accurate in vitro tests to exclude hypersensitivity. A radioallergosorbent test (RAST) is a potentially good supplementary approach, but there is little information on the suitability of this method to diagnose penicillin hypersensitivity in subjects with a negative skin test to benzylpenicillin. METHODS: A total of 133 patients with a negative skin test to benzylpenicillin G (PG) and all of whom developed allergic reactions to PG were studied. RAST was used to detect eight kinds of specific IgE antibodies to penicillins in serum, which included four kinds of major and minor antigenic determinants to four penicillin drugs. The combination sites for the specific IgE antibodies were studied by RAST inhibition test. RESULTS: The rate of positive reactions for the specific IgE antibodies was 59.40% (79/133). Of the eight kinds of antigenic determinants, the positive rates for specific IgE against the major and minor determinants were 39.10% (52) and 42.86% (57) respectively. Of the four drugs, positive cases only to PG were 10 (7.5%), were significantly fewer than the cross-reacting positive cases (36) to PG (P < 0.01). In the RAST inhibition studies all drugs exhibited good inhibitory potencies, and in some instances the side-chain of the penicillins could induce specific responses with a variable degree of cross-reactivity among the different penicillins. CONCLUSION: Radioallergosorbent test is a good complementary test in persons who are skin-test negative with PG, and the sensitivity of RAST increases with increasing specificity of IgE antibodies to be detected. 6-APA and the groups, making part of the different side-chains on penicillins, all contributed to the cross-reactivity.

The specificity of tests for anti-beta-lactam IgE antibodies declines progressively with increase of total serum IgE.

BACKGROUND: Immediate allergic reactions to beta-lactam antibiotics are mediated by specific IgE antibodies. The Phadia CAP System FEIA is a commercial method for quantification of specific IgE. We wished to determine anti-beta-lactam IgE antibodies in patients without penicillin allergy but with high levels of total IgE. METHODS: Sera from 41 patients (31 with high total IgE, 10 with low total IgE) were analyzed for IgE antibodies specific to penicilloyl G, penicilloyl V, amoxicilloyl and ampicilloyl using the CAP FEIA((R)) method that was available up to 2006. Seven sera that tested positive were rechecked in a new improved system available after 2006. RESULTS: In patients without a history of penicillin allergy, the specificities of commercial tests for anti-beta-lactam IgE antibodies were 100%, 60%, 27% and 20% at total IgE levels of 8-263 kU/l, 500-664 kU/l, 1000-2000 kU/l and > 2000 kU/l, respectively. In seven retested sera, only 2 (28%) were still positive for penicillin-specific IgE antibody. CONCLUSION: Before 2006, tests for anti-beta-lactam IgE antibody in patients with total IgE > 500 kU/l were probably often false positive. Patients who were diagnosed as penicillin allergic before 2006 solely on the basis of a positive CAP FEIA test for specific IgE should be considered for diagnostic reevaluation.

Management of tolerance induction in patients with suspected penicillin allergy--a case study.

BACKGROUND: In some diseases penicillin is the treatment of choice. Case studies have shown a good response for the treatment of circumscribed scleroderma or scleroderma adultorum of Buschke. A suspected allergy to penicillin in a patient's history may limit this helpful therapy option. Allergy testing is often inconclusive. If indicated, tolerance induction leading to therapy with penicillin can be carried out. PATIENTS AND METHODS: We present two patients with circumscribed sclerosis and scleredema Buschke, who had a suspected allergy to penicillin. Due to limited therapy options and in insufficient response to other therapeutics, the decision for a tolerance induction with penicillin was made. Penicillin was successfully administered by following a scheme of tolerance induction starting with oral doses and ending with high doses of intravenous penicillin G. RESULTS: In both cases, penicillin G, administered over a period of three weeks, was well tolerated up to the high dose of 3 x 10 Mega IU/day. Substantial clinical improvement was achieved in all cases without any complications. CONCLUSION: This case study demonstrates that a suspected allergy to penicillin does not preclude an eventual treatment with this valuable drug. Allergy testing should routinely be carried out first. If suspicion of an allergy persists, tolerance induction can be attempted according to the new scheme described here. Starting with a careful, initial oral dose regimen, treatment can be continued with an increasing intravenous dose followed by maintenance therapy with high-dose penicillin G. It should be clear that this policy is only restricted for patients who are at risk for a hypersensitivity to penicillin, i.e., because of a clinical manifested incompatibility in the past.