Mary E. von Blomberg

CD4+CD25hi regulatory T‐cell frequency correlates with persistence of human papillomavirus type 16 and T helper cell responses in patients with cervical intraepithelial neoplasia

CD4+CD25hiCTLA4+FoxP3+ regulatory T cells (Treg) have been shown to maintain immune tolerance against self antigens and increased circulating frequencies have been reported in various types of cancers. Circulating invariant natural killer T‐cells (iNKT) are reduced in cancer patients and low iNKT frequency is related to poor prognosis. It is not yet clear whether high Treg numbers and low iNKT cell numbers pose an increased risk for the progression of premalignant lesions or whether Treg and iNKT cell numbers are influenced by dysplasia. We therefore studied prospectively the relation between iNKT cell and Treg frequencies and the natural course of human papillomavirus type 16 (HPV16) induced pre‐malignant cervical dysplasia in 82 patients who participated in a nonintervention cohort study of women with abnormal cytology. Treg frequencies were significantly increased in women who had persistent HPV16 infection. Within the HPV16 persistence group there was no difference in Treg frequencies among patients who developed a CIN3 lesion and patients who did not progress to CIN3. Furthermore, Treg frequencies were increased in patients who had detectable HPV16 E7 specific IL‐2 producing T‐helper cells, which suggests a causal role of HPV infection in Treg development in parallel with HPV16 specific T helper cells. No evidence was found for a role for iNKT cells in persistence of HPV16 and progression of HPV16 induced CIN lesions. However, HPV‐persistence‐associated Tregs may explain the inefficacy of concomitant persistence associated immunity and may contribute to subsequent progression to neoplasia.

Improved detection of allergen-specific T-cell responses in allergic contact dermatitis through the addition of ‘cytokine cocktails’

Abstract:  The gold standard for the diagnosis of allergic hypersensitivity is skin patch testing with the suspected allergens. This diagnostic tool, however, has distinct disadvantages, and therefore the development of alternative or complementary in vitro tests is of great importance. In this study, we evaluate the applicability of an in vitro test method, as developed earlier for nickel allergy, to detect allergen‐specific T cells in the blood of patients allergic to frequent sensitizers (chromate, cobalt, paraphenylenediamine, fragrances and chloromethyl‐isothiazolinone). Peripheral blood mononuclear cells (PBMCs) of allergic patients and healthy controls were cultured in the absence or presence of allergen. Additionally, type 1 (IL‐7 and IL‐12) or type 2 (IL‐7 and IL‐4) stimulating cytokines were added; after 6‐day proliferation, IFN‐γ and IL‐5 secretions were determined. Without the addition of cytokines, consistent allergen‐induced proliferation was observed in PBMCs of nickel‐allergic patients only. By contrast, the addition of type 1 or type 2 stimulating cytokines resulted in a significantly enhanced allergen‐specific proliferation for all allergens tested (sensitivity increased from 26 to 43% or 38%, respectively, P < 0.05). In these cultures, allergen‐induced IFN‐γ and IL‐5 secretion was also significantly increased, compared to healthy controls (P < 0.05, for IFN‐γ sensitivity 79%, specificity 93%; for IL‐5 sensitivity 74%, specificity 81%). In conclusion, these results demonstrate an increased proliferative capacity and cytokine production by allergen‐specific T cells from allergic patients, but not of healthy individuals upon stimulation with allergens in combination with type 1 or 2 skewing cytokines. The present data warrant further exploration of the application of this test to a broader set of allergens.

Successful Induction of Allergic Contact Dermatitis to Mercury and Chromium in Mice

Availability of reproducible mouse models for allergic contact dermatitis (ACD) to the metal allergens nickel, mercury and chromium, would be of great value for pathogenetic and preventive studies. We explored epicutaneous sensitization to nickel, mercury and chromium in mice in which oral grooming of the sensitization site was prevented by a plaster cast around the abdomen and lower thorax. This procedure was based on earlier findings that oral ingestion of allergen could prevent contact sensitization. The present results show that BALB/c mice can be readily sensitized to mercury and chromium using this epicutaneous casting method, without the further use of adjuvants. With nickel, however, neither this method, nor conventional methods involving the use of Freunds complete adjuvant (FCA) were effective.

Development of a concomitant nickel and chromium sensitization model in the guinea pig

Although nickel allergy is the most frequent contact hypersensitivity in man, reports on successful nickel sensitization in experimental animals are scarce. Chromium hypersensitivity, on the other hand, is readily induced in guinea pigs. In this study we set out to obtain reproducible nickel sensitization in guinea pigs, in order to establish an animal model for immunospecific tolerance and desensitization studies in which two non-cross-reacting metal allergens, chromium and nickel, could be studied simultaneously. Strong and reproducible sensitization to nickel was achieved by injecting low amounts of Freunds complete adjuvant and nickel sulfate in a split-adjuvant procedure. Strong erythematous reactions were observed as early as 14 days after sensitization and could be elicited both by intradermal and open epicutaneous challenges. Optimal evaluation was with nickel sulfate administered epicutaneously in 40% dimethyl sulfoxide to enhance skin penetration. Hypersensitivity could be transferred with lymphocytes and not with serum. Sensitization procedures for nickel and chromium then could successfully be combined in a double sensitization procedure. With four different guinea pig strains no genetic restriction was observed for the induction of nickel or chromium sensitivity. However, for both metals a clear sex and age dependence was observed: female guinea pigs reached a higher degree of sensitization than males, whereas sensitization in young animals was relatively weak.

Mononuclear phagocyte function in head and neck cancer. Chemotactic responsiveness of blood monocytes in correlation between histologic grade of the tumor and infiltration of these cells into the tumor area

The chemotactic responsiveness of peripheral monocytes and the acid‐phosphatase activity of tumor‐infiltrating macrophages, as well as the ultrastructural appearance, were studied in 40 patients with squamous cell carcinoma of the head and neck. The chemotactic responsiveness was found to be decreased in carcinoma patients, and this value appeared to be positively correlated in individual patients with the number of tumor‐infiltrating macrophages, as well as with the histologic grade of the tumor. Patients with poorly differentiated malignancies showed impaired monocyte chemotactic responsiveness and low numbers of tumor‐infiltrating macrophages. Macrophages present in the parenchyma of the tumor showed a weak and diffuse pattern of acid phosphatase activity. The acid phosphatase activity of stromal macrophages was much stronger and distributed in foci. Electron microscopic examination of the parenchymal macrophages revealed low numbers of lysosomes and the presence of tumor cell debris in the cytoplasm of the cell, without any sign of a surrounding phagosomal membrane. Together with the weak cytochemical reactivity, this probably indicates the poor functional state of the phagocyte when infiltrated in the parenchyma of the tumor. Low molecular weight factors derived from the tumor are known to decrease chemotactic responsiveness of peripheral monocytes. The poor functional state of the macrophages infiltrated within tumor parenchyma might be explained by assuming that a high concentration of such factors in the near vicinity of malignant cells causes toxic effects in macrophages.

Differential Capacity of Macrophages from Various Sources to Act as Hapten-Specific Stimulator Cells in vitro

Lymphocytes from 2,4-dinitrochlorobenzene, contact sensitized guinea pigs show increased DNA synthesis in vitro when stimulated by dinitrophenylated macrophages. In this study, macrophage-containing cell suspensions were isolated from various sources (spleen, lungs, peritoneal cavity) and from the peritoneal cavity also after different stimuli (oil, thioglycollate, starch, lymphokines). These cells were then haptenized and investigated on their capacity to act as stimulator cells in vitro. In addition, a possible relationship between Ia-positivity of the hapten-presenting cell suspensions and the induction of DNA synthesis was studied. The results demonstrate (1) that the hapten-presenting capacity of macrophages differs with respect to the source and the way of elicitation, (2) that oil-induced peritoneal exudate cells are the most suitable stimulator cells for in vitro assays in contact sensitivity, and (3) that the cellular expression of Ia antigens is in itself no warrant for hapten-presenting capacity.

Effects of Contact Sensitization and Delayed Hypersensitivity Reactions on Immune Responses to Non-Related Antigens

Guinea pigs were immunized intracutaneously into the ears with sheep red blood cells (SRBC). Application of a sensitizing dose of the contact allergen dinitrochlorobenzene (DNCB) onto the same ears was shown to suppress or enhance the humoral response to SRBC depending on the time of application. When guinea pigs were sensitized to a contact allergen, application of a sensitizing dose of a non-related allergen on the same ears either had no effect or caused a clear enhancement of the development of delayed type hypersensitivity (DTH). Strongest enhancement was found when both sensitizations were performed on the same day. Further experiments on the effects of a concomitant DTH reaction elicited at the site of application of a contact allergen showed a strong potentiation of DTH when B-cell suppression was minimized by pretreatment with cyclophosphamide (CY). It was considered that CY-DTH-immunopotentiation might be a useful tool for achieving a higher level of sensitivity after epicutaneous sensitization.