Reduction of intestinal carcinogen absorption by carcinogen-specific secretory immunity.

A secretory immune response to the carcinogen 2-acetylaminofluorene (AAF) was elicited in rabbits by directly immunizing the small intestine with an AAF-cholera toxin conjugate. High-titer, high-affinity secretory immunoglobulin A (IgA) antibody to AAF was secreted into the intestinal lumen in response to this immunogen. Immune secretions reduced the transepithelial absorption of a 125I-labeled derivative of AAF by more than half. This reduction of absorption by hapten-specific IgA suggests that oral vaccines against carcinogens and toxicants could be developed for humans.

The enteric immune response to shigella antigens.

Mucosal immunity to some enteropathogens occurs naturally following infection. By learning how to optimize initiation of the mucosal immune response it will be possible to develop vaccines against a wide variety of enteropathogens and their toxic products. In the past few years, we have examined stimulation of the mucosal response to Shigella antigens. We have found that the mucosal memory response to Shigella LPS can be stimulated by oral immunization with live, but not with killed Shigella. This primes specific B lymphocytes which, following rechallenge, quickly migrate from the Peyer's patches to mesenteric lymph nodes, the spleen, and back to the Peyer's patches. We have found that the uptake of S. flexneri is the initial step in developing a mucosal immune response to Shigella. Whereas there is little difference between the initial uptake of virulent and avirulent bacteria by M cells, pathogenic strains of Shigella are able to replicate following their uptake by the specialized M cells located in the follicle-associated epithelium of the gut. This likely serves as the source of the ulcerative lesions found in dysentery. Lastly, we have detected a vigorous secretory IgA response to Shiga toxin. The titer of IgA activity to Shiga toxin from these loop secretions correlated well with the ability to prevent Shiga toxin cytotoxin effects in vitro. The extremely vigorous mucosal immune response to Shiga toxin makes this an attractive alternative to cholera toxin to potentiate the secretory IgA immune response.

Demonstration of M cells in the specialized follicle-associated epithelium overlying isolated lymphoid follicles in the gut.

Within the epithelium that overlies the dome regions of Peyer's patches, exist specialized surface epithelial cells (M) which function to take up macromolecules from the gut lumen. These cells may be of great importance in processing antigenic material in the gut. The predominant lymphoid structures of the small intestine are isolated lymphoid follicles, by virtue of their frequency. These follicles are difficult to study because they are not grossly visible. In the present study, three guinea pigs drank India ink mixed into their water for 1, 3, and 5 months. Two hours prior to sacrifice, animals were given an intraintestinal injection of ferritin or India ink. Using a hand lens, the Peyer's patches and isolated follicles were clearly identified among the villi of the intestine. Light microscopy revealed ink in the surface epithelium covering the isolated follicles and within the substance of the follicles. Transmission electron microscopy demonstrated M cells over isolated follicles and Peyer's patches. These cells had lighter staining cytoplasm, while the mitochrondria stained darker with prominent cristae, and the microvilli were shorter. Therefore, M cells do exist within isolated follicles and structurally appear the same as those found in Peyer's patches. This implicates the isolated follicles in the overall antigen processing role of gut-associated lymphoid tissues. The present method facilitates identification of isolated lymphoid follicles which will allow functional studies to be performed on these structures.

MicroELISA method for measurement of human serum thymosin alpha 1.

A microELISA for the estimation of human serum thymosin alpha 1 is described. In this assay, antibody to thymosin alpha 1 is pre-incubated with the standard or serum at 4 degrees C. Unbound antibody in the liquid-phase then binds with solid-phase thymosin alpha 1. The method is sufficiently sensitive for measuring serum levels of thymosin alpha 1 and highly reproducible. The serum levels measured with the microELISA are comparable to serum levels of thymosin alpha 1 determined by the previously described radioimmunoassay for thymosin alpha 1.

Measurement of thymosin alpha 1 by disassociation microELISA.

A disassociation microELISA was devised for the estimation of thymosin alpha 1, a chemically characterized thymic polypeptide isolated from bovine thymosin fraction 5. Antiserum to synthetic thymosin alpha 1 was raised in rabbits. Thymosin alpha 1 in liquid phase competed with a solid-phase-bound thymosin alpha 1 for this highly specific antibody. The method is specific, sensitive, reproducible and capable of detecting as little as 100 pg/ml of thymosin alpha 1.

A rapid and sensitive screening method for the detection of anti-2-acetylaminofluorene immunoglobulins.

A method is described in which anti-2-acetylaminofluorene immunoglobulins may be detected using a simple and sensitive screening procedure. The method is based on immunoglobulin binding of an 125I derivatized 2-aminofluorene radiotracer. Tracer binding is not isotype specific, and thus the method is useful for the detection of either IgG or IgA. Competitive binding experiments with the radiotracer were used to determine the specificity of immunoglobulin response by measurement of cross-reactivity with related ligands. This method allows quantitation of the immune response to the carcinogen in serum and other biological fluids (i.e., intestinal secretions).

Arizona hinshawii osteomyelitis with antecedent enteric fever and sepsis. A case report with a review of the literature.

A case of Arizona osteomyelitis of the spine which occurred 11 months after an episode of gastroenteritis and enteric fever is presented. As close biochemical and antigenic relative of Salmonella, Arizona infection produces a similar clinical course with gastrointestinal manifestations frequently preceding localized infections by several months. The boney lesion in the present case and in three of the four other cases of Arizona osteomyelitis described in the literature was a chronic inflammation which may have a xanthomatous component. The bone destruction caused by Arizona infection is less severe than that of tuberculosis or pyogenic osteomyelitis. Proposed treatment of Arizona osteomyelitis consists of debridement of the localized infection and long term antimicrobial therapy.

Intestinal mucosal immune defense mechanisms.

The intestinal mucosal immune defense mechanisms involve both humoral and cellular immunity. The prominence of suppressor/cytotoxic T lymphocytes in the epithelial layer suggests that these interepithelial lymphocytes play a role in defense against infections within this layer. Secretory IgA is overwhelmingly the major humoral immune response along the gastrointestinal tract and along other mucosal surfaces (respiratory tract, mammary glands, salivary glands, and lacrimal glands). While the functions of secretory IgA are incompletely understood, it is clear that it prevents attachment of microorganisms and toxins (cholera toxin, shiga toxin, etc.) to the surface epithelial cells. Furthermore, secretory IgA may collaborate with eosinophils or killer lymphocytes to mediate cytotoxic reactions against enteropathogens. By learning more about the mucosal immune response, we should be able to understand the relationship between the lamina propria plasmacytosis in inflammatory bowel disease and the increased number of interepithelial lymphocytes that we see in gluten-sensitive enteropathy and the underlying pathogenic mechanisms.

Detection of immunoglobulin A by immunofluorescence in glycol methacrylate-embedded human colon.

Several methods to demonstrate human immunoglobulin (Ig) A in glycol methacrylate (GMA)-embedded colon are explored. Following routine fixation in neutral buffered formalin, the tissue was dehydrated and embedded in GMA. Two-micron thick sections were heat-fixed (70 to 80 degrees C) to glass slides and then soaked in either xylene or acetone to etch the GMA. Following rehydration of the sections in serial alcohols and water, tissues were treated with phosphate buffered saline (PBS) and/or a variety of enzymes (trypsin, protease V, protease VI, pepsin, and papain) for varying periods of time. After an overnight soak in PBS the tissues were stained with fluorescein-conjugated anti-human IgA and then rinsed with PBS. This technique demonstrated excellent fluorescence of the IgA-containing plasma cells and of the discrete IgA-containing vesicles normally found in the surface epithelial cells of colon. Results were best with tissues etched with xylene and digested with 0.25 mg/ml protease V in PBS, pH 7.4, for 2 hr at 37 degrees C, followed by an overnight soak in PBS. Almost no fluorescence was seen in sections not digested with enzymes. The present method offers a simple convenient technique to perform immunofluorescence for immunoglobulin antigens on GMA-embedded tissue.

Correlation of histopathologic evidence of disease activity with the presence of immunoglobulin-containing cells in the colons of patients with inflammatory bowel disease.

Immunofluorescence of formalin-fixed, paraffin-embedded tissues was performed to study the plasma cell population in 114 colonic specimens from 58 patients. Correlation of the histopathologic stage of disease activity with the isotypes and numbers of immunoglobulin-containing cells in the lamina propria demonstrated highly significant (P less than 0.001) increases in the mean numbers of IgG- (18-fold), IgA- (twofold) and IgM- (sixfold) containing cells in specimens from patients with active inflammatory bowel disease as compared with control specimens. Increased numbers of immunoglobulin-containing cells were uncommon in inactive inflammatory bowel disease and in reactive mucosa. No deposition of immunoglobulin-containing immune complexes was found at any stage of disease activity. These findings suggest that immune complex-mediated damage does not play a major role in the epithelial damage in inflammatory bowel disease. In future studies, it will be of importance to determine whether the antibody from immunoglobulin-containing cells seen in patients with inflammatory bowel disease can effect damage via an antibody-dependent cell-mediated cytotoxicity mechanism.

The effects of heparin on lipoproteins in high-resolution electrophoresis of serum.

Sera from heparinized patients commonly display anodal slurring of both the alpha and beta lipoproteins when they are examined by high-resolution electrophoresis (HRE). In this study, the authors examined the effect of heparin and lipoprotein lipase on the electrophoretic migration of alpha and beta lipoproteins in sera. The addition of 10 to 1,000 units of heparin/mL to normal sera resulted in a concentration-dependent anodal slurring of the beta lipoproteins. At 40 units/mL, the beta lipoprotein band was not visible when the Paragon blue protein stain was used. The beta lipoprotein could be seen as a wide, faintly staining band with lipoprotein stain. The alpha lipoprotein band on the same gel was unaffected by the added heparin. High-resolution electrophoresis of other sera from patients who were therapeutically heparinized demonstrated anodal slurring of both alpha and beta lipoproteins independent of heparin concentration. Immunofixation electrophoresis (IFE) studies confirmed that apolipoproteins A and B were slurred within their respective bands. Heparin activates lipoprotein lipase with release of free fatty acids (FFA) from very low density lipoproteins and chylomicrons. To test this effect on migration, sera were incubated with lipoprotein lipase in vitro. The anodal slurring of both the alpha and beta lipoprotein was associated with the amount of FFA production. Individuals interpreting electrophoretic patterns should be aware that both the alpha and beta lipoproteins can migrate and slur anodally in heparinized patients. In addition, when the beta lipoprotein band interferes with the identification of monoclonal gammopathies, its migration can be selectively altered by the addition of 40 units/mL heparin to the sample.

The effects of exogenous free fatty acids on lipoprotein migration in serum high-resolution electrophoresis: addition of free fatty acids improves visualization of normal and abnormal alpha1-antitrypsin.

Abnormalities in the alpha1 region are occasionally difficult to interpret on high-resolution electrophoresis (HRE) gels because alpha1-antitrypsin (A1AT) can be obscured by elevated levels of lipoprotein. The addition of saturated free fatty acids (SFFAs) to serum samples causes a selective anodal migration of lipoproteins when examined by HRE. This "clearing" of the alpha1 region improves visualization of A1AT. In this study, we evaluated 6- to 24-carbon SFFAs for their ability to cause anodal migration of serum lipoproteins; identified the optimal SFFA and determined its effects on other proteins; and applied the SFFA to problematic serum samples, including those containing dense alpha lipoprotein regions. When added to serum samples, a mixture of medium-chain (12-18 carbon) SFFAs caused a dose-dependent anodal migration of the alpha and beta lipoproteins and albumin. Lauric acid (C(12:0); 3.2-6.5 mmol/L) caused an optimal effect--maximal anodal migration of alpha lipoproteins and clearing of the alpha1 region, facilitating inspection of that area for A1AT variants. At the optimal C(12:0) concentration, the migration and resolution of A1AT were minimally affected, anodal slurring of beta lipoproteins and albumin were slight, and there was no effect on interpretation of monoclonal proteins. At higher concentrations, C(12:0) increased anodal migration of beta lipoproteins and decreased the resolution of A1AT. C(12:0) at 3.2 mmol/L in serum samples of heparinized patients showed an exaggerated effect similar to higher concentrations of C(12:0) in nonheparinized serum samples. Hyperbilirubinemia did not alter the effect of C(12:0) on serum proteins. C(12:0) treatment of serum samples displaying dense alpha lipoprotein bands on HRE dramatically improved visualization of A1AT. We report the incidental identification of A1AT variants in two such samples treated with C(12:0). The addition of C(12:0) to serum samples markedly improves visualization of normal and abnormal A1AT in the alpha1 region in HRE.

Current practices in clinical flow cytometry. A practice survey by the American Society of Clinical Pathologists.

The American Society of Clinical Pathologists surveyed 136 laboratories actively engaged in performing clinical flow cytometric testing to determine the demographics of these laboratories, the credentials of the personnel involved with testing, the volume and types of tests performed, and how data are analyzed and interpreted. These results are reported with commentary based on previous surveys and recommended practice guidelines.

Densitometric scanning of high-resolution electrophoresis of serum: methodology and clinical application.

The recent introduction of high-resolution electrophoresis into many clinical laboratories has expanded the information available about protein abnormalities. While most laboratories interpret high-resolution electrophoresis patterns by direct visual examination of the stained electrophoresis strips, much useful information can be attained by supplemental densitometric scanning of these preparations. In the present report, we detail a method to perform densitometric scanning of high-resolution electrophoresis strips, and compare these results to those performed by standard cellulose acetate electrophoresis. Further, we evaluate the quantitative information by comparing the results from the densitometer scans with nephelometric quantification of specific monoclonal proteins. The present method gives a correlation coefficient of 0.97 when the gamma region densitometric scan is compared to the quantification of IgG by nephelometry in patients with known gamma-migrating IgG monoclonal gammopathies. Further, twofold dilution studies of these specimens showed excellent linearity. By providing objective, reliable, quantitative information, densitometric scanning of high-resolution electrophoresis strips is a useful adjunct to direct visual examination of these specimens.

A modification of the automated immune precipitin method for quantitation of human serum immunoglobulins.

The use of the automated immune precipitin method to measure human serum immunoglobulins has been reported by several groups. The authors encountered difficulties in evaluating the technic even when they incorporated such recent modifications as the use of polyethylene glycol to enhance the reaction. By altering the flow pattern and prediluting each specimen, they were able to increase sampling rate, decrease processing time, and decrease the frequency of line obstructions while obtaining reproducible standard curves without using polyethylene glycol. There was good correlation between the results obtained by their modification of the technic and those obtained by the Fahey radial immunodiffusion method. As with other automated immune precipitin systems, abnormally high values gave characteristic notched peaks and lipemic specimens were centrifuged in order to obtain the infranatant for quantitation. The present modification of the automated immune precipitin system provides a rapid, simple and efficient method to quantitate human serum immunoglobulins.

Low maternal serum unconjugated estriol during prenatal screening as an indication of placental steroid sulfatase deficiency and X-linked ichthyosis.

Placental sulfatase deficiency is an X-linked metabolic defect that occurs in about 1 in 2,000 to 5,000 males. It is associated with congenital ichthyosis. In this report, the authors document a case of placental sulfatase deficiency detected during routine prenatal screening of maternal serum by the triple test: serum alpha-fetoprotein (AFP), unconjugated estriol (uE3), and human chorionic gonadotropin (hCG). At 16-weeks gestation, her AFP was 20.9 IU/mL (multiple of the median [MOM] 0.83), hCG was 14.4 mIU/L (MOM 0.42) and her uE3 was 0.01 nmol/L (MOM 0.01). The extremely low uE3 indicated a possible placental sulfatase deficiency, congenital adrenal hypoplasia, or other unknown abnormality. On receiving this information, the obstetrician obtained a family history that was consistent with ichthyosis in the maternal grandfather and his siblings. Biochemical analysis of placenta documented the lack of sulfatase activity. This case illustrates that an extremely low level of maternal uE3 should prompt investigation of the family for evidence of X-linked ichthyosis associated with placental sulfatase deficiency.

Coagulation disorders in patients with monoclonal gammopathies.

Monoclonal gammopathies can present as disorders of coagulation whether the gammopathy is due to a malignant B-cell lymphoproliferative disorder or a monoclonal gammopathy of undetermined significance. Detection of the monoclonal gammopathy involves quantification of immunoglobulins including kappa and lambda along with high-resolution electrophoresis of serum. Immunofixation is occasionally needed on serum and always needed on urine for the final diagnosis.

Immunoglobulins from the sera of immunologically activated persons with pairs of electrophoretic restricted bands show a greater tendency to aggregate than normal.

From in vitro data, it has been speculated that pairs of endogenous restricted bands migrating in close proximity in the gamma region upon high resolution serum electrophoresis (HRE) represent circulating immune complexes (CIC). Using a polyethylene glycol (PEG) method to separate CIC, we found a very high correlation between the presence of such band pairs and elevated levels of CIC (CHI2 = 25.7, p less than 0.001) in 51 sera. HRE appears to be a good screening technique to identify, with a high degree of certainty, samples with elevated levels of CIC for delineation by more specific methods. Yet, examination by gel-filtration chromatography and precipitation with PEG indicated that the molecules comprising the band patterns were not CIC, but polyclonal 7S IgG. These bands are usually found in patients with chronic activation of the immune system. Fractionation of the sera from 5 such patients with various cuts of PEG indicated that the average IgG concentration in the 2.5-5%, and 5-7.5% cuts from patients was 3.99 g/l and 2.2 g/l, while from healthy subjects the concentrations were 0.68 g/l and 2.88 g/l. This reversed precipitation pattern was seen both for absolute levels of IgG and for percent of total IgG. On the average the amount of precipitation of IgG in the 2.5-5% fraction of patients was about 5-fold above that seen in the healthy subjects. The endogenous bands were not associated with any specific cut of PEG, but appeared to be proportionally distributed in accord with the levels of IgG. The data is consistent with the idea that immunologically activated patients exhibit a greater tendency for immunoglobulins to associate than normal. This propensity to aggregate may cause CIC to form in situ in local compartments even though CIC do not appear to be present upon analysis by biochemical techniques.

Selective induction of mucosal immune responses to 2-acetylaminofluorene.

Mucosal vaccination with chemical carcinogens coupled to enterotoxins such as cholera toxin (CT) can elicit carcinogen-specific immunoglobulin secretion into the intestinal lumen. The present study examines the ability of several related bacterial enterotoxins and their subunits to act as adjuvants or carrier proteins in stimulating an intestinal secretory IgA (S-IgA) response to 2-acetylaminofluorene (AAF). Using Thiry-Vella loops in rabbits, CT, cholera toxin B subunit (CTB) and the recombinant B subunit of the heat labile enterotoxin from E. coli (rLTB) were all found to be effective carrier proteins and adjuvants for eliciting S-IgA anti-AAF. However, marked differences in the ratio of mucosal S-IgA to serum IgG production were observed. CT elicited the highest luminal S-IgA anti-AAF titers as well as the highest ratio of intestinal S-IgA/serum IgG when used as an adjuvant. Conversely, rLTB elicited a high serum IgG anti-AAF titer but only a modest intestinal S-IgA response. Dialysis studies using monoclonal IgA versus IgG anti-AAF on opposing sides of a semipermeable membrane demonstrated the potential importance of the intestinal S-IgA/serum IgG ratio. A high "intestinal" IgA/"serum" IgG ratio abolished carcinogen transfer to the "serum" side of the membrane, while a low ratio enhanced transfer. Thus, to generate an active mucosal immune response capable of blocking carcinogen absorption, the carrier protein or adjuvant should be selected to optimize the intestinal S-IgA/serum IgG ratio.

Autoreactivity and altered immune responses in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a poorly understood condition that is associated with a wide variety of immunologic alterations. Because its pathogenesis is unknown, these immunologic alterations have been investigated with an eye toward unraveling the complex mechanism of injury in the bowels of these patients. There are several lines of evidence suggesting that IBD is related to immunologic events. The histopathology of active disease resembles the Arthus reaction, whereas the presence of antiepithelial cell antibodies is reminiscent of Goodpasture's disease. Antibodies against many microorganisms and autoantibodies to mucosal components are commonly found in these patients. Further, there is a marked increase in plasma cells in the lamina propria of patients with active IBD. It is important to keep these findings in perspective. No studies to date have been able to determine whether the features are entirely primary events, that is, related to the initial damage to the intestinal mucosa. If the surface mucosa is injured by an as-yet-unidentified agent, the immunologic findings in IBD may be secondary events. Nonetheless, the similarity in histopathology of the experimental immunologic models of IBD to the human disease encourages investigators to pursue the etiology of this complex disease.