The influence of CO2 versus helium insufflation or the abdominal wall lifting technique on the systemic immune response.

BACKGROUND: Both laparoscopic and conventional surgery result in activation of the systemic immune response; however, the influence of the laparoscopic approach, using CO2 insufflation, is significantly less. Little is known about the influence of alternative methods for performing laparoscopy, such as helium insufflation and the abdominal wall lifting technique (AWLT), and the systemic immune response. METHODS: Thirty-three patients scheduled for elective cholecystectomy were randomly assigned to undergo laparoscopy using either CO2 or helium for abdominal insufflation or laparoscopy using only the AWLT. The postoperative inflammatory response was assessed by measuring the white blood cell count, C-reactive protein (CRP) and interleukin-6 (IL-6). The postoperative immune response was assessed by measuring monocyte HLA-DR expression. RESULTS: CRP levels were significantly higher 1 day after helium insufflation when compared with CO2 insufflation; however, no differences were observed 2 days after surgery. The AWLT resulted in significantly higher levels of CRP both 1 and 2 days after surgery when compared with either CO2 or helium insufflation. A small increase in postoperative IL-6 levels was observed in all groups, but no significant differences were seen between the groups. After both helium insufflation and AWLT a significant decrease in HLA-DR expression was observed, in contrast to the CO2 group. CONCLUSION: Carbon dioxide used for abdominal insufflation seems to limit the postoperative inflammatory response and to preserve parameters reflecting the immune status. These findings may be of importance in determining the preferred method of laparoscopy in oncologic surgery.

Incipient ovarian failure and premature ovarian failure show the same immunological profile.

PROBLEM: Incipient ovarian failure (IOF) is characterized by regular menstrual cycles, infertility and a raised early-follicular FSH in women under 40. IOF might be a precursor or a mitigated form of premature ovarian failure (POF). Disturbances in the immune system may play a role in ovarian failure. METHOD OF STUDY: Autoantibodies and lymphocyte subsets were determined in 63 POF patients, 50 IOF patients, and 27 controls. RESULTS: The prevalence of autoantibodies did not differ between the groups. There was a statistically significant difference in lymphocyte subsets between the control group and the POF group, with the IOF group taking an intermediate position. We found a decrease in percentage of T-suppressor cells with a rise in T-helper/T-suppressor cell ratio, a decrease in natural killer cells, and an increase in B lymphocytes and HLA-DR positive T cells. CONCLUSIONS: These data support the concept that IOF is a mitigated form of POF. The question remains whether these changes are the cause or the consequence of the ovarian failure.

Should relatives of coeliacs with mild clinical complaints undergo a small-bowel biopsy despite negative serology?

BACKGROUND AND OBJECTIVES: Small intestinal lesions in coeliac disease (CD) have a variable severity. Early diagnosis of CD is important because treatment allows a normal psycho-physical development, especially in children, and can avoid associated disorders. The aim of this study was to evaluate the predictive value of screening parameters for the detection and estimation of CD prevalence in first-degree relatives. METHODS: The screening was performed in 338 first-degree relatives of 134 coeliac families. Questionnaires and a physical examination followed by haematological analyses and serologyfor IgA anti-endomysium (EMA)/IgA antigliadin (AGA) antibodies were used in orderto selectthe candidates for small-bowel biopsy. The small-bowel biopsy was indicated on the basis of clinical complaints, laboratory tests and serology performed in 96 (28%) of the study group. RESULTS: CD was diagnosed in 17/96 cases. Six of the 17 showed total villous atrophy (VA) (Marsh IIIc), five subtotal VA (Marsh IIIb) and six partial VA (Marsh IIIa). EMA and AGA were strongly positive in the six patients whose intestinal biopsy showed total VA. However, only one coeliac out of the six patients with partial VA had positive EMA and AGA. CONCLUSION: A significant proportion of coeliacs may be missed if cases are screened by serology only. Although endomysial antibody assay has been reported as a highly sensitive and specific test for detection of CD, we argue that using only EMA and AGA in screening is not enough for investigation of the true prevalence of CD. A combination of clinical parameters as described in this study and laboratory/serological tests is an important and practical contribution to improving the detection rate of CD.

Reversal of orally induced T-cell tolerance by subcutaneous administration of interleukin-12 at the site of attempted sensitization.

Feeding of proteins causes peripheral T-cell tolerance, as revealed by reduced delayed-type hypersensitivity (DTH) reactivity after immunization. Using ovalbumin-fed mice, we studied whether putatively immunostimulatory cytokines could reverse this state of mucosal tolerance. It was found that local administration of neither IL-2, IFN-gamma, nor GM-CSF resulted in reversal of tolerance. In contrast, subcutaneous administration of IL-12 at the site of attempted immunization resulted in complete recovery of DTH reactivity. The dichotomy between the two Th1-stimulatory cytokines IFN-gamma and IL-12 was also reflected by different effects on ovalbumin-specific antibody isotypes. Although both IFN-gamma and IL-12 downregulated serum IgG1-levels in tolerant mice, suggesting decreased ovalbumin-specific Th2 function, only local administration of IL-12 led to increased serum Th1-related IgG2a levels. These results support the view that potentiation of Th1 effector function is critical for reversal of mucosal tolerance.

Cross-reactivity of human nickel-reactive T-lymphocyte clones with copper and palladium.

Twenty Ni-reactive T-lymphocyte clones were obtained from eight different donors and analyzed for their ability to cross-react with other metals. All Ni-reactive T-lymphocyte clones were CD4+CD8- and recognized Ni in association with either HLA-DR or -DQ molecules. Based on the periodic table of the elements, the metals Cr, Fe, Co, Cu, and Zn from the same horizontal row as Ni, and Pd and Pt from the same vertical row, were selected to study T-lymphocyte clone cross-reactivity. Distinct cross-reactivity patterns were found that could be divided into three major groups: Ni-reactive T-lymphocyte clones i) cross-reacting with Cu, ii) cross-reacting with Pd, or iii) without cross-reactivity. Major histocompatibility complex class II-restriction patterns of Cu- and Pd-induced proliferative responses did not differ from those for the Ni-induced responses. In vitro cross-reactivities with Cu and Pd may be favored by their bivalency and location next to Ni in the periodic table, and the similarity of these metals to Ni in binding to histidine residues of peptides in the pocket of major histocompatibility complex class II molecules. The present findings suggest that Cu and Pd hypersensitivities, which are occasionally observed in Ni-allergic patients, may be due to cross-reactivities at the T-cell clonal level rather than to concomitant sensitization.

Oral induction of tolerance to nickel sensitization in mice.

Antigen contact via the alimentary tract prior to sensitization may result in systemic immunologic unresponsiveness ("oral tolerance"). The induction of oral tolerance seems an attractive strategy to combat undesired immune responses, such as allograft rejection and autoimmune and allergic diseases. We describe clear and reproducible sensitization to nickel in mice reared under nickel-free conditions. Hypersensitivity was induced by injecting nickel sulfate intradermally into the flank skin and elicited by injecting the metal salt into the pinnae of the ears. The effectiveness of orally induced hyporesponsiveness could be inferred from a low degree of hypersensitivity obtained with mice raised and maintained in cages with nickel-releasing covers and water nipples. This mouse model for the assay of nickel hypersensitivity was used for oral tolerance studies by administrating non-toxic doses of nickel sulfate in drinking water or intragastrically prior to sensitization. In these animals, the development of delayed-type hypersensitivity was suppressed in a dose-dependent way, and the hyporesponsiveness could be transferred by CD8+ cells. The antigen specificity of this oral tolerance could be demonstrated by the concomitant use of sensitization and challenge procedures for nickel and chromium. The hypersensitivity assay described provides a versatile, highly reproducible experimental model to study immunoregulation of oral tolerance to clinically relevant metal allergens.

Persistent immune tolerance to nickel and chromium by oral administration prior to cutaneous sensitization.

Oral administration of allergens, foreign proteins, or cell-bound antigens may induce systemic suppression of subsequent humoral and cell-mediated immune responses ("oral tolerance"). The induction of specific immune tolerance provides a potential strategy for treatment of T-cell-dependent immune diseases. Therefore, in depth studies into preconditions for optimal and persistent tolerance induction are mandatory. Here we report on such studies in a guinea pig model using the non-cross-reactive contact allergens nickel and chromium. Feeding per os of nickel sulfate or potassium dichromate did not trigger systemic TDTH-effector functions. Instead, short feeding periods led to a dose-dependent, and metal-specific, suppression of subsequently induced allergic contact hypersensitivity. Administration of the allergens onto the oral mucosa was most effective in the induction of immune tolerance. When first sensitizing attempts were delayed until 1 year after feeding, the degree of unresponsiveness was reduced. In contrast, with cutaneous contacts starting shortly after the feeding period, tolerance was fully stable and undiminished for at least 2 years. Thus, in orally treated guinea pigs cutaneous contacts provide boosting tolerogenic signals, supporting the view that oral tolerance does not result from clonal deletion but from active antigen-specific immunosuppression. Indeed, unresponsiveness to cutaneous immunization could be transferred by lymphoid cells from fed guinea pigs in a metal-specific way.

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.

Induction of immunological tolerance by oral administration of nickel and chromium.

Oral administration of nickel-chromium to guinea pigs by way of a fixed occlusal splint, or the incorporation of metallic powder or salts into the pelleted food, did not induce hypersensitivity to these metals. In addition, a subsequent attempt to immunize the pre-treated guinea pigs failed in most animals, whereas non-pre-treated guinea pigs became clearly hypersensitive. These results show that oral administration of nickel and chromium induced a state of (partial) tolerance to both metals.

Induction of immunological memory in the skin. Role of local T cell retention.

Using an experimental contact sensitivity model in guinea-pigs, evidence is presented that hapten (DNCB or oxazolone) specific T lymphocytes may persist for several months in previous sites of inflammation. Immunological memory, revealed by accelerated contact skin reactions upon retesting with the hapten, was limited to the original contact skin reaction sites. This 'local skin memory' to DNCB or oxazolone could be induced in both specific and non-specific skin inflammatory reactions, provided the animals had been sensitized to the hapten not longer than 2 weeks before. In animals which had been sensitized more than 1 month earlier, local skin memory could be induced if the animals received a booster application of hapten shortly (0-2 days) before primary skin testing. From these results we conclude that recently activated T cells may enter inflammatory sites non-specifically, producing specific local immunological memory. This memory may last several months. Accumulation of hapten specific T cells at inflammatory sites may be important in retest reactivity, in flare-up reactivity and in chronic inflammation.

Mononuclear phagocyte function in head and neck cancer: NBT-dye reduction, maturation and migration of peripheral blood monocytes.

The number of blood monocytes, their ability to mature into macrophages, their NBT-dye reduction capacity and their migration towards casein were studied in 29 patients with squamous cell carcinoma of the larynx, 12 patients with squamous cell carcinoma at other sites within the head and neck and two groups of male controls, one of 29.7 yrs. +/- 7.2 (SD) and the other of 71.4 yrs +/- 6.8 (SD). An age dependency of monocyte function in healthy individuals was established. Increased numbers of monocytes per ml blood were found in the older men. These cells showed an enhanced migration toward casein. Their maturation capacity was decreased. A clear impairment of migration toward casein was found in the two groups of carcinoma patients. The number of monocytes was only marginally affected. The maturation was found to be enhanced. An impaired recruitment of mononuclear phagocytes at the site of malignant growth due to diminished migratory capacities might be important for failing immune surveillance.

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.

Influence of anti-inflammatory drugs on the interaction of lymphocytes and macrophages.

The helper function of macrophages in lymphocyte stimulation is well known, but there are indications that macrophages may also exert a suppressor effect on lymphocytes. This effect might be due to prostaglandins secreted by the macrophages. In order to test this hypothesis anti-inflammatory drugs, some of which are known inhibitors of prostaglandin synthesis, were added to a series of phytohaemagglutinin (PHA)-stimulated lymphocyte cultures containing different proportions of macrophages and lymphocytes. The experiments showed that high concentrations of all drugs were inhibitory. Moderate concentrations of some of the PG-synthesis-inhibiting drugs (like indomethacin and mefenamic acid), however, appeared to have a stimulatory effect. The stimulation was more pronounced in cultures containing a high proportion of macrophages. These results support the assumption that macrophages release prostaglandins, which suppress PHA-induced lymphocyte proliferation.

Hapten-cell conjugates in DNCB contact sensitivity. In vitro stimulation with DNP-conjugates optimally inducing contact sensitivity in vivo.

Until now in vitro stimulation tests have failed to detect DNCB contact sensitivity in guinea-pigs sensitized epicutaneously without the use of adjuvants. In this study DNP-conjugates optimally inducing contact sensitivity in vivo were tested for their capacity to detect DNCB contact sensitivity in vitro in a lymphocyte transformation assay. In vivo contact sensitivity measurements in guinea-pigs which had been immunized with different hapten-cell conjugates, showed that (1) living cells should be used for conjugation with the allergen, (2) macrophages are optimal carriers and (3) syngeneity is not required. Therefore, DNFB-coated peritoneal macrophages (viable when conjugated) were used as an antigen for in vitro stimulation. Using this conjugate, a highly reproducible antigen-specific increase in DNA synthesis could be obtained in lymph node lymphocytes from guinea-pigs that had been sensitized to DNCB by epicutaneous application of the allergen without the use of adjuvants.

A simple cytochemical method for distinguishing EAC rosettes formed by lymphocytes and monocytes.

A simple cytochemical method for EAC rosettes in suspension is described which allows an easy distinction between rosettes formed by lymphocytes and monocytes. The method is based on the fact that monocytes, in contrast to lymphocytes, possess a specific cytoplasmic esterase.

Interference of simultaneous skin tests in delayed hypersensitivity.

The interference of two simultaneous skin test reactions of intermediate strength has been studied in the guinea-pig, using four different antigens, i.e. ovalbumin, horse cytochrome c, PPD and oxazolone. Skin test reactions were evaluated at 4, 24 and 48 h by measuring three parameters: increase in skin thickness, diameter of erythema and intensity of erythema. When an Arthus reaction was elicited simultaneously with a delayed hypersensitivity (DH) reaction, no effect on the DH reaction was observed. When two simultaneous DH reactions were elicited with different antigens, the risk of interference appeared to be rather small. When, however, the same antigen was used for both skin tests, suppression of at least one parameter of a DH-reaction was found in almost all experiments. Suppression of one skin test by another one could not be reduced by introducing a large distance between the two skin tests. As complete inhibition of either of the parameters never occurred, multiple skin testing may allow one to obtain a qualitative impression of the state of delayed hypersensitivity; when, however, reliable quantitative data are needed, the performance of more than one skin test at a time should be avoided.

Effects of contact sensitization and delayed hypersensitivity reactions on immune responses to non-related antigens. Modulation of Immune responses.

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.

Direct macrophage migration inhibition test in DNCB contact sensitized guinea pigs.

Using dinitrochlorbenzene contact-sensitized guinea pigs, several DNP conjugates have been assayed in the direct macrophage migration inhibition test (MMIT). Although no significant differences could be observed between the carriers used, conjugates prepared from serum proteins and epidermal extracts tended to give the strongest inhibition of macrophage migration. Conjugates prepared from cells cultured in vitro in the presence of hapten did not cause more inhibition than control conjugates prepared in the absence of cell metabolism. The direct MMIT showed statistical differences between sensitized and nonsensitized groups of guinea pigs. However, none of the conjugates permitted conclusions to be drawn with regard to individual animals.