Tissue proteolysis in appendicitis with perforation.

BACKGROUND: Matrix metalloproteinases (MMPs) and serine proteases are able to degrade the extracellular matrix (ECM) and modulate immune responses in the gastrointestinal tract. The purpose of this study was to investigate local proteolysis in perforated appendicitis and its association with the appendix perforation. MATERIALS AND METHODS: Biopsies were taken at the sites of perforation (n = 15) and with a gradually increased distance from it. The expression and distribution of MMP-1, -2, and -9, the tissue inhibitor of metalloproteinases type (TIMP-1), plasminogen activator inhibitor type1 (PAI-1), and urokinase plasminogen activator (uPA) were measured by ELISA. The distribution of MMP-9, TIMP-1, uPA, and PAI-1 in perforated, nonperforated, and uninflamed appendix was investigated by immunohistochemistry with monoclonal antibody technique. RESULTS: MMP-1 expression was highest close to the perforation and was gradually decreased in biopsies in more distal locations (P < 0.01). MMP-9 showed a similar pattern being highest at the sites of perforation (P < 0.05), while MMP-2 expression showed a trend in the opposite direction without statistically significance. The expression of TIMP-1 trended lower at the sites of perforation. PAI-1 was highest at the sites of perforation (P < 0.01) and the uPA expression was similarly elevated close to and at the perforation. CONCLUSIONS: These data indicate a key role of MMP in the pathogenesis of appendix perforation. A local imbalance between MMP-9 and the inhibitor TIMP-1 could potentially contribute to the tissue injury leading to an appendix perforation. The overexpression of PAI-1 at the sites of perforation may also contribute to tissue damage.

Immunohistochemical analysis of granzyme B in non-perforated appendicitis--short communication.

The aim of the present study was to determine the expression ofgranzyme B in non-perforated appendicitis. Appendix biopsies were obtained from the patients with clinically diagnosed as acute appendicitis and subjects admitted for elective abdominal surgery. All biopsies from the patients were non-perforated and histologically divided into acute and non-acute appendicitis. Granzyme B expression was assessed immunohistochemically. The results showed that granzyme B expression in both acute and non-acute appendicitis was significantly lower than that in the control appendix tissues (P < 0.05). The expression of this cytotoxic protein in acute and non-acute appendicitis was comparable (P > 0.05). Therefore, the results of the present study suggest that reduced expression of granzyme B in non-perforated appendicitis may reflect low cytotoxic activities which may prevent tissue damage.

Terminal deoxynucleotidyl transferase-positive cells in spleen, appendix and branchial cleft cysts in pediatric patients.

We evaluated spleens (n = 26), appendices (n = 10) and branchial cleft cysts (n = 6) for TdT-positive cells in pediatric patients. In spleen, appendix and branchial cleft cysts the range of TdT-positivity was 0-13, 0-96 and 0-6 TdT+ cells/hpf, respectively. In spleens, scattered TdT+ cells were seen most frequently in periarteriolar lymphoid sheath regions.

Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix.

Studies in mouse, human, and chicken suggest that activation-induced deaminase (AID) is involved in three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class-switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B cell maturation events. We report here successful cloning of rabbit AID and isolation of AID protein from rabbit appendix-cell nuclear and cytoplasmic extracts. We succeeded in identifying and locating AID protein in cells by immunohistochemical and immunofluorescent staining techniques and examined colocalization of AID and other molecules important for Ab diversification. This report extends our knowledge about AID to a mammalian species that uses gene conversion to diversify rearranged Ig genes. Although much work remains to understand fully the mechanism of action of AID and its association with other cellular components, the rabbit system now offers a particularly useful model for future studies of these dynamics.

Expression of 5-lipoxygenase in specialized epithelial cells of nasopharyngeal-associated lymphoid tissue.

Leukotrienes are lipid mediators that are produced primarily by certain types of leukocytes. The synthesis of the leukotriene LTB(4) is initiated by the enzyme 5-lipoxygenase and completed by LTA(4) hydrolase. Epithelial cells constitutively express LTA(4) hydrolase but normally lack 5-lipoxygenase. In this study, we report that the stratified squamous epithelial cells from inflamed or hyperplastic tissues of palatine and pharyngeal tonsils (nasopharyngeal-associated lymphoid tissue) express 5-lipoxygenase protein. The localization of 5-lipoxygenase was indicated by immunohistochemical staining and presence confirmed by immunoblot. Positive staining for 5-lipoxygenase in infiltrating leukocytes in inflamed tissues served as internal positive controls for immunohistochemical staining. Staining for 5-lipoxygenase in appendix tissue was negative for epithelial cells while positive for polymorphonuclear leukocytes, indicating that 5-lipoxygenase expression is not a general feature of epithelial cells in mucosa-associated lymphoid tissue. In tonsils, 5-lipoxygenase staining was pronounced in broad regions but reduced or absent in others, suggesting regional regulation of expression. Epithelial cells of tonsils were also positive for 5-lipoxygenase activating protein and leukotriene A(4) hydrolase, indicating a capacity to produce LTB(4). Taken together, these results suggest that the specialized epithelial cells of the mucosa-associated lymphoid tissue of human tonsils can synthesize LTB(4). This lipid mediator may serve to modulate the function of cells within the lymphoid tissue as well as promote an inflammatory response.

Nitrergic hyperinnervation in appendicitis and in appendices histologically classified as normal.

CONTEXT: The pathogenesis of appendicitis remains poorly understood. Despite new diagnostic techniques, appendices removed from patients with suspected appendicitis often appear histologically normal on conventional examination. There is increasing evidence of involvement of the enteric nervous system in immune regulation and in inflammatory responses in the gastrointestinal system. OBJECTIVE: To investigate the nitrergic innervation of (a) acutely inflamed appendices, (b) appendices classified as histologically normal from patients with a clinical diagnosis of appendicitis, and (c) normal control appendix specimens, using the whole-mount preparation technique. PATIENTS AND DESIGN: Full-thickness specimens were collected from 28 acutely inflamed appendices (age range, 3.2-13.4 years), 31 histologically normal appendices removed from patients (age range, 5.7-13.6 years) with suspected appendicitis, and 23 histologically normal appendices from patients (age range, newborn to 12.1 years) undergoing elective abdominal surgery (controls). Whole-mount preparation using nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and neuronal nitric oxide synthase immunohistochemistry were performed. The density of myenteric plexus was measured with a computerized analysis system. RESULTS: The density of myenteric plexus in normal appendix specimens was similar to that of large bowel from the newborn period up to 3 years of age; this density decreased significantly thereafter. The myenteric plexus of normal appendix specimens from patients older than 4 years demonstrated smaller ganglia connected by thin nerve bundles, compared to larger ganglia and nerve bundles in large bowel. Significant neuronal hypertrophy was found in 55% of acutely inflamed and 41% of histologically classified normal appendix specimens. The myenteric plexus of these appendix specimens had even thicker nerve bundles connecting an increased number of ganglion cells. CONCLUSIONS: Differences in the architecture of the myenteric plexus in patients older than 3 years suggest an altered function and motility of appendix in the early years of life. The significant increase in neuronal components of the myenteric plexus in a high proportion of acutely inflamed and histologically normal appendix specimens is unlikely to have developed during a single acute inflammatory episode. This suggests an underlying chronic abnormality as a secondary response to chronic luminal obstruction or repeated inflammatory episodes in the histologically normal appendix.

Changes of protein gene product 9.5 (PGP 9.5) immunoreactive nerves in inflamed appendix.

The existence of chronic appendicitis is controversial. In this prospective study, we investigated possible changes in the innervation of the appendix under different pathological conditions and correlated histological findings with clinical observation. Thirty appendectomy specimens and 14 appendices obtained from organ donors or patients who underwent right hemicolectomy were immediately fixed in Bouin's solution and processed for immunocytochemistry using an antiserum directed against the panneuronal marker protein gene product 9.5 (PGP 9.5). The density of PGP 9.5 immunostaining was evaluated by digitized morphometry. Significant differences in the density of the PGP 9.5-immunoreactive area were detected in the mucosal layer. In the nonacute appendicitis group, PGP 9.5 was increased (10.99 +/- 3.15%) as compared to acute appendicitis (3.89 +/- 1.77%) and controls (4.98 +/- 1.25%). The significant increase of PGP 9.5 in nonacute appendicitis may suggest axonal sprouting leading to hyperinnervation of the mucosa. This may be a neuronal factor in the pathophysiology of the disease and pain symptoms.

Immunocytochemical localization of aromatic L-amino acid decarboxylase in human, rat, and mouse bronchopulmonary and gastrointestinal endocrine cells.

Aromatic L-amino acid decarboxylase (AADC) catalyzes the cellular decarboxylation of L-aromatic amino acids and is therefore involved in the synthesis of several biogenic amines. Application of the indirect immunoperoxidase method on human, rat, and mouse tissues using specific antibodies to AADC revealed all AADC-containing cells. Besides mast cells and adrenergic nerve fibers, the following cells were immunostained: neuroendocrine cells in the tracheobronchial epithelium; neuroepithelial bodies in the bronchopulmonary epithelium; Kultschitzky cells in the small intestine and appendix as well as adrenal chromaffin cells. All the latter cells belong to the so-called APUD system, the "D" in the acronym standing for the activity of the enzyme aromatic L-amino acid decarboxylase. Immunocytochemistry for AADC may become an additional tool not only to highlight APUD cells in tissue sections but also to differentiate the sites of cellular amine synthesis from those of amine storage.

Diamine oxidase as a marker of intestinal integrity in acute appendicitis.

In the operative treatment of appendicitis the so called negative appendectomy is an important issue because of its increased morbidity. From the hypothesis that the intestinal diamine oxidase activity is a suitable marker of mucosal integrity, the distribution pattern of the enzyme in appendices histologically classified as inflamed or not inflamed was studied. Histologically apparent inflammation of the appendix was connected with a significant reduction of diamine oxidase activity. The determination of this enzymic activity may be a simple and sensitive test for mucosal inflammation of the appendix even at a very early state. This could reduce the rate of negative appendectomies and influence thereby risk-cost-benefit calculations.

Histochemical enzymatic assessment of the human appendix at different ages.

The appendectomy specimens submitted to surgical pathology exhibit a wide variety of morphological appearances. We have evaluated the activities of acid phosphatase, alpha-naphthyl esterase, and leucineaminopeptidase in 100 human appendixes divided into six age groups in order to ascertain whether any histochemical pattern was related to the age group rather than to pathological condition. Our findings support that the submucosal fibrosis is unrelated to the patient's age, as well as appendiceal obliteration. It is noteworthy that the mucosa of the appendix may retain its lymphoid constituents at any age and that young subjects may already show appearances of involution up to lumen obliteration.

[Enzymatic reaction of the follicular cell population in the human appendix]. / Reazioni enzimatiche della popolazione follicolare in appendici umane.

Histochemical techniques were employed to determine acid phosphatase and alpha-naphthylesterase activities in follicular cells from the mucosa of surgically removed human appendixes. These very reactive cells are found in two areas: in the middle of the light centre and in the surroundings of this centre. The former are globose, arranged with a certain degree of order, and have a reticular anchorage that does not appear to be particularly affected by the physiological and pathological events that occur in the appendix, whereas those of the border vary in number or may even be absent. They have an elongated shape and are also anchored in a reticulum that in this case is more consistent. The result is that in certain forms of appendiceal disease the pale centre of the follicle is closed in a kind of shell formed of very reactive cells. The identification and significance of these two groups of cells of the light centre are discussed.

Enzymatic activation of lymphoid population following inflammatory reactions in the human appendix.

We have histochemically evaluated the LAP, AP and ANE activities of the human appendix in 100 surgical specimens. The mucosa has been particularly investigated for its different aspects during inflammatory conditions. Two main facts have been ascertained: the subepithelial location of macrophages, and the AP and ANE activities of two groups of cells in the germinal centers. Subepithelial macrophages show conspicuous AP activity that correlates with the degree of inflammatory response and of exposure to luminal antigens. In the germinal centers, AP and ANE activities disclose two further groups of positive cells, probably the same cells reacting to both technics. One group is rooted to the core of the germinal center; the other to the intermediate area between the germinal center and the follicular mantle.

Human carbonic anhydrase isoenzyme C. Effects of some fixatives on the antigenicity and improvements in the method of localization.

The effects of some alcohol and aldehyde containing fixatives on the antigenicity of human carbonic anhydrase isoenzyme C (HCA C) were tested in order to reveal the most suitable method for the immunohistochemical localization of this enzyme. The use of 2% and 4% paraformaldehyde or 2% glutaraldehyde solutions before immunoperoxidase (PAP) staining resulted in the loss of HCA C-specific reactivity in the surface epithelial cells of human appendicular and gastric mucosae, whereas the antigenic reactivity of HCA C was well retained in the parietal cells of gastric glands. In corresponding tissue sections fixed with one of the alcohol containing solutions (abs. methanol, methanol + chloroform 2:1 or Carnoy fluid) both the surface epithelial and parietal cells showed HCA C-specific immunostaining after the PAP procedure. In addition, the antigenicity of HCA C was found to be well preserved in some tubular cells of human kidney fixed in Carnoy fluid. The paraffin infiltration at relatively low temperature did not markedly affect the enzyme antigenicity. Fixation in Carnoy fluid coupled with paraffin embedding at 55-60 degrees C in vacuo was found to give the best preservation of the antigenicity of HCA C with good morphological integrity for light microscopical localization.

Lymphocyte positions in the dome epithelium of the rabbit appendix.

The apico-basal distribution of lymphocytes within the epithelium covering the domes of lymphatic tissue in the wall of the rabbit appendix was investigated in single and serial sections stained either for general histology, for cytoplasmic basophilia and acidophilia, or for nonspecific esterase activity. From the base to the summit of a dome, four zones numbered proximo-distally 1-4 were distinguished. Epithelial cells migrate from base to summit, as indicated by mitotic figures in zone 1, the gradual change from cytoplasmic basophilia to acidophilia in zones 2 to 4, and visible extrusion of cells from zone 4 at the summit. Zone 1 was free of lymphocytes. Most of the lymphocytes in zone 2 were intercellular and randomly arranged, but a few in this zone were within tapered epithelial cells modified by a process extending basally to the basement membrane. Small numbers of these tapered epithelial cells also occurred in zone 3. The large clusters of ten to 12 lymphocytes that characterized zone 3 were intercellular and impinged the apical regions of epithelial cells. Serial sections at the level of the distal cluster of zone 3 showed lymphocytes located also more basally, and some of these lymphocytes appeared to be passing through the basement membrane back into the lymphoid tissue of the dome. Epithelium of zone 4 over the distal surface of a dome was largely free of lymphocytes. Apparently most infiltrating lymphocytes form intercellular clusters and then return to the subepithelial lymphatic tissue.

[Lymphoid tissue of the human vermiform appendix and its cellular composition at different ages]. / Limfoidnaia tkan' cherveobraznogo otrostka cheloveka i ee kletochnyi sostav v razlichnye vozrastnye periody.

The lymphoid tissue of 55 human appendices has been studied beginning from 25 weeks of the intrauterine development up to 80 years of age. Most of lymphocytes have been found to include immunoglobulines of various types; in some cases alcaline phosphatase activity was evident in lymphocytes of the mantle zone of lymphatic follicles in children and grown-up persons, and lack of acidic phosphatase activity in lymphocytes make it possible to consider the appendicular lymphoid tissue as a B-dependent type of lymphoid tissue. A suggestion is made that during the intrauterine development and the early childhood the appendicular lymphocytes could settle in other lymphoid formations.

Immunohistochemical localization of human carbonic anhydrase isoenzyme C.

Methods for immunohistochemical localization of human carbonic anhydrase isoenzyme C (HCA C) with indirect fluorescent antibody and immunoperoxidase techniques are described. Both methods revealed large amounts of this "high activity" isoenzyme in the mucosae of human stomach and appendix. With the indirect immunofluorescent method the presence of the enzyme in human erythrocyte cytoplasm was also demonstrated. Correlations of present findings with those obtained with the traditional histochemical methods for demonstration of carbonic anhydrase activity are discussed.

An unusual case of phospholipidosis.

We present the results of a structural, histochemical and lipid-chromatographic study of tissues obtained at postmortem from an unusual case of phospholipidosis. A previous biopsy of the appendix and liver (Elleder et al., 1975a) had revealed a predominance of phosphoglyceride storage, principally of lysobisphosphatidic acid (LBPA) postmortem material showed that this lipid was stored exclusively in central neurons. In the spleen and the lymph node, however, sphingomyelin (SP) was shown, histochemically and chromatographically, to be the main lipid stored. Total sphingomyelinase (SPase) activity in the appendix was reduced to about 50% of normal. Neuroaxonal dystrophy (NAD) and a conspicuous discrepancy between the degree of distension of some neurons and their lipid content deserve special mention. The case is contrasted with classical sphingomyelinosis; the complexity of the Niemann-Pick group of diseases is discussed as an indication of the difficulties of classification of any atypical case.