Peroxidase Immunocytochemistry for the Detection of Specific Anti-Hapten (Penicilloyl) Antibody Producing Cells in the Spleen, After Injection of a Hapten-Carrier Conjugate

Cells producing specific antibodies against the hapten penicilloyl (Pen) are demonstrated in tissue sections of the spleen, using peroxidase immunocytochemistry. Horseradish peroxidase-human serum albumin-penicilloyl (HRP-HSA-Pen) conjugate, prepared by coupling Pen to HRP-HSA conjugate, was used for detection of anti-Pen producing cells in the spleen after intravenous injection of bovine gamma globulin-penicilloyl (BGG-Pen) conjugate. HRP-HSA conjugate was used as a control to exclude the possibility that positive cells contained antibody against a common determinant in both HSA and BGG. The method may be applied for the detection of cells producing antibodies against other happens as well.     

Peroxidase immunocytochemistry for the detection of specific anti-hapten (penicilloyl) antibody producing cells in the spleen, after injection of a hapten-carrier conjugate

Cells producing specific antibodies against the hapten penicilloyl (Pen) are demonstrated in tissue sections of the spleen, using peroxidase immunocytochemistry. Horseradish peroxidase-human serum albumin-penicilloyl (HRP-HSA-Pen) conjugate, prepared by coupling Pen to HRP-HSA conjugate, was used for detection of anti-Pen producing cells in the spleen after intravenous injection of bovine gamma globulin-penicilloyl (BGG-Pen) conjugate. HRP-HSA conjugate was used as a control to exclude the possibility that positive cells contained antibody against a common determinant in both HSA and BGG. The method may be applied for the detection of cells producing antibodies against other haptens as well.     

A rapid and simple hapten conjugation method for monoclonal antibodies to be used in immunoenzyme single and double staining procedures

A rapid and simple method was developed for the haptenization of monoclonal antibodies (MAbs), to be used in immunoenzyme single and double staining techniques. Using this method minute amounts of MAbs can be haptenized without purification of the antibody or removal of the excess hapten. The haptenized MAb can be ready for use with 2.5 h. The method consists of a direct incubation of the antibody with the haptenizing agent and subsequent addition of an amino acid solution to stop the reaction. Commonly available reagents were tested, of which dinitrofluorobenzene, trinitrobenzene sulfonic acid and an oxazolone derivative gave the best results. The procedure was evaluated by using MAbs directed against lymphoid cell surface membrane antigens in an indirect immunoenzyme staining on frozen sections using peroxidase or alkaline phosphatase-conjugated anti-hapten antibodies as second step antibody. It was found that those MAbs, which show good staining results in conventional indirect immunoenzyme procedures, can also be used successfully after haptenization in single as well as double staining procedures. Combination of haptenized and biotynilated MAbs gave good results when weakly reactive MAbs had to be included in immunoenzyme double staining.     

Penicillin Hypersensitivity

An enzyme-linked immunosorbent assay for the detection of anti-pencillin antibodies of the several Ig classes is described. The results of the ELISA in 350 sera of patients suspected of penicillin hypersensitivity are compared with those of the haemagglutination test. In 105 sera penicillin-specific IgM and/or IgG was demonstrated with the ELISA, the HA test being positive in 49 of these 105 sera. However , in another 14 sera IgM anti-penicillin antibodies could be shown only with the HA. In 10 sera penicillin-specific IgE was demonstrated with the ELISA, only four of these being also positive with the RAST. IgE was always found in combination with IgG and/or IgM. The positive correlation of the ELISA and the RAST with the intracutaneous test on penicillolypolylysine was 26.9% and 15.4% respectively. The ELISA is a simple and reproducible method for the detection of anti-penicillin antibodies, being more sensitive than the HA and the RAST.     

High sensitivity and negative predictive value of sentinel lymph node biopsy in a retrospective early stage oral cavity cancer cohort in the Northern Netherlands

Objectives

In cT1‐2N0, oral squamous cell carcinoma (OSCC) occult metastases are detected in 23%‐37% of cases. Sentinel lymph node biopsy (SLNB) was introduced in head and neck cancer as a minimally invasive alternative for an elective neck dissection in neck staging. Meta‐analyses of SLNB accuracy show heterogeneity in the existing studies for reference standards, imaging techniques and pathological examination. The aim of this study was to assess the sensitivity and negative predictive value (NPV) of the SLNB in detecting occult metastases in cT1‐2N0 OSCC in a well‐defined cohort.

Design

Retrospective study. The SLNB procedure consisted of lymphoscintigraphy, SPECT/CT‐scanning and gamma probe detection. Routine follow‐up was the reference standard for the SLNB negative neck. Histopathological examination of sentinel lymph nodes (SLN) consisted of step serial sectioning, haematoxylin‐eosin and cytokeratin AE1/3 staining.

Setting

Two comprehensive oncology centres.

Participants

A total of 91 consecutive patients with primary cT1‐2N0 OSCC treated by primary resection and neck staging by SLNB procedure between 2008 and 2016.

Main outcome measures

Sensitivity and negative predictive value.

Results

In all cases, SLNs were harvested. A total of 25 (27%) patients had tumour‐positive SLNs. The median follow‐up was 32 months (range 2‐104). Four patients were diagnosed with an isolated regional recurrence in the SLNB negative neck side resulting in an 85% sensitivity and a 94% NPV.

Conclusion

In our cohort, the SLNB detected occult metastases in early OSCC with 85% sensitivity and 94% NPV. This supports that SLNB is a reliable procedure for surgical staging of the neck in case of oral cT1‐2N0 SCC.     

The contact allergen dinitrochlorobenzene (DNCB) and respiratory allergy in the Th2-prone Brown Norway rat

All LMW respiratory allergens known to date can also induce skin allergy in test animals. The question here was if in turn skin allergens can induce allergy in the respiratory tract. Respiratory allergy was tested in Th2-prone Brown Norway (BN) rats by dermal sensitization with the contact allergen dinitrochlorobenzene (DNCB; 1%, day 0; 0.5%, day 7) and a head/nose-only inhalation challenge of 27mg/m3 of DNCB (15 min, day 21), using a protocol that successfully identified chemical respiratory allergens. Skin allergy to DNCB was examined in BN rats and Th1-prone Wistar rats in a local lymph node assay followed by a topical patch challenge of 0.1% DNCB. Sensitization of BN rats via the skin induced DNCB-specific IgG in serum, but not in all animals, and an increased number of CD4+ cells in the lung parenchyma. Subsequent inhalation challenge with DNCB did not provoke apneas or allergic inflammation (signs of respiratory allergy) in the BN rats. However, microarray analysis of mRNA isolated from the lung revealed upregulation of the genes for Ccl2 (MCP-1), Ccl4 (MIP-1beta), Ccl7 and Ccl17. Skin challenge induced considerably less skin irritation and allergic dermatitis in the BN rat than in the Wistar rat. In conclusion, the Th2-prone BN rat appeared less sensitive to DNCB than the Wistar rat; nevertheless, DNCB induced allergic inflammation in the skin of BN rats but even a relatively high challenge concentration did not induce allergy in the respiratory tract, although genes associated with allergy were upregulated in lung tissue.     

Increased CCL27-CCR10 expression in allergic contact dermatitis: implications for local skin memory

Allergic contact dermatitis (ACD) is a T-cell-mediated disease in which expression of a distinct repertoire of chemokines results in the recruitment of effector T cells into the skin. While it is becoming clear which chemokines and receptors determine the development of ACD, the mechanisms involved in the retention of T cells in the skin after resolution of inflammation are still unknown. Unravelling these mechanisms will help us to understand local skin memory as observed in retest reactivity and flare-up reactions. This study was designed to evaluate the role of chemokine-chemokine receptor interactions in local T-cell retention. The results show that expression of the CCR10 targeting ligand CCL27 is not only increased during inflammation, but also remains increased several weeks after clinical responsiveness to patch testing. In parallel with increased CCL27 expression, an increased number of infiltrating cells could still be detected in skin that, clinically, had returned to normal 21 days after patch testing. These persisting cells were characterized as CD4+ cells expressing CCR10, while no CD8+ CCR10+ cells could be detected. The presence of these cells is most likely an allergen-mediated effect, as increased levels of CCL27 and CCR10 could not be detected 21 days after initiating an irritant contact dermatitis reaction. In contrast to CCL27, increased expression of CXCL9, CXCL10, and CXCL11 could only be observed during the clinically inflammatory phase of ACD. In conclusion, local CCL27-mediated retention of CCR10+ CD4+ T cells in sites previously challenged by ACD could be responsible for phenomena such as local skin memory observed in retest reactions and flare-up reactions in which the presence of persisting T cells results in an accelerated inflammatory response upon renewed allergen challenge.     

Identification of anti-inflammatory drugs according to their capacity to suppress type-1 and type-2 T cell profiles

BACKGROUND: Down-regulation or modulation of T cell activity by immunosuppressive drugs is an effective treatment in diseases where exaggerated T cell responses play a role. A primary effect of the anti-inflammatory drugs (AIDs) is inhibition of the synthesis of growth factors, such as IL-2, thereby down-regulating T cell proliferation. However, it is still largely unknown to what extent these AIDs are able to down-regulate specifically type-1 or type-2 T cell cytokine production, and whether they can down-modulate chemokine receptor expression, thereby preventing migration of T cells to the site of inflammation. OBJECTIVE: We investigated the suppressive effect of dermatologically used AID (cyclosporin A (CsA), lactoferrin (LF), 1 alpha, 25-dihydroxyvitamin D(3) (VD(3)), hydrocortisone (HC), di-methyl-fumarate (DMF), diclofenac (DF)) on both type-1 and type-2 T cells. Since allergic contact dermatitis is a skin disorder in which an exaggerated T cell response of both types of T cell subsets can be observed, we used this disorder as a model to study the capacity of AID to suppress type-1 or type-2 T cell responses. METHODS: Peripheral blood mononuclear cells of nickel allergic patients were cultured in the presence of allergen and increasing concentrations of AID. Proliferation was determined by measuring (3)H thymidine incorporation; chemokine receptor (CCR10, CCR4, CXCR3) expression was studied by flow cytometric analysis and IFN-gamma or IL-5 cytokine production was measured by ELISA. RESULTS: Three major patterns can be distinguished regarding the effect of AID on T cell responses. The first group, including CsA and LF, inhibited non-selectively T cell proliferation, chemokine receptor expression and cytokine production, with CsA as the most potent drug tested. A second group of AID, which included VD(3), HC and DMF, suppressed mainly type-1 T cell responses, as revealed by strong interference with IFN-gamma production and CXCR3 expression, and limited effects on either or both IL-5 and CCR4 expression. The third pattern was displayed by DF, which down-regulated IL-5 production and CCR4 expression, whereas IFN-gamma and CXCR3 were unaltered. CONCLUSIONS: Using a contact allergy model, we have demonstrated that various AIDs show distinct pharmacological profiles in that either type-1 or type-2 or both T cell responses are suppressed. These results should contribute to a more rational selection of AID in treating inflammatory skin diseases mediated by either or both of these T cell subsets.     

Detection of specific anti-hapten antibody-producing hybridoma cells in tissue sections of spleen and liver by hapten-enzyme conjugates using an immunoenzyme approach

A method is presented for the detection of hybridoma cell growth in vivo by making use of the specific antigen (hapten) recognition properties of the produced monoclonal antibodies. Horseradish peroxidase (HRP)-labeled antigen was used for demonstration of intracellular specific antibodies in hybridoma cells. It appeared that intravenously injected anti-penicilloyl-producing hybridoma cells developed mainly in the red pulp of the spleen; to a lesser extent they were found in the liver.