David F. Keren

Immunohistological characterization of intraepithelial and lamina propria lymphocytes in control ileum and colon and in inflammatory bowel disease

Using monoclonal antibodies to T and B lymphocytes, to natural killer cells, and to HLA-DR antigen, we characterized the lymphocyte population within the epithelial and lamina propria regions in control intestine and colon, and in grossly involved and in grossly uninvolved intestine and colon of patients with active inflammatory bowel disease. There were significantly more intraepithelial T cells in control ileum than in control colon. In comparison to control, there was a heterogeneity of alterations in intraepithelial and lamina propria T lymphocyte subsets (T11+, T8+, T4+) in inflammatory bowel disease. B lymphocytes were not detected within the lamina propria, except when found in and adjacent to lymphoid aggregates. Leu 7+ cells were uncommon in the lamina propria of control ileum and colon and in diseased tissues. The majority of intraepithelial lymphocytes did not express HLA-DR. Epithelial cells of control colon did not express HLA-DR while epithelial cells of control ileal tissues and of diseased colonic and ileal specimens expressed HLA-DR antigen. Only small numbers of lamina propria T cells expressed HLA-DR in both control and disease tissues. There was intense expression of HLA-DR by monocytes and modest expression of HLA-DR by capillary and lymphatic endothelial cells. The induction of HLA-DR expression by diseased colonic epithelium and the observation that lymphatic endothelium expresses HLA-DR are new observations, and we established that Leu 7+ cells are present in very small numbers in both normal and diseased intestine and colon.

Guidelines for Clinical and Laboratory Evaluation of Patients With Monoclonal Gammopathies

This guideline provides the recommendations of an expert panel for the clinical and laboratory evaluation of patients suspected of having a clinical condition that produces a monoclonal protein in serum or urine. The recommendations describe the clinical conditions in which a monoclonal protein should be sought, the optimal sequence of testing to diagnose and monitor these patients, and the most effective laboratory procedures.

Procedures for the evaluation of monoclonal immunoglobulins

A wide variety of techniques are available for the screening, characterization, and quantification of monoclonal proteins. These techniques vary in regard to the expense, skill and intensity of labor involved, and sensitivity for detection of low levels of monoclonal proteins or of those with unusual migration. Detection of monoclonal proteins requires the use of high-resolution electrophoresis (either gel-based or capillary) and immunofixation (or immunosubtraction). Immunoelectrophoresis is not recommended. Urine for detection of monoclonal free light chains should be from 24-hour samples, and the aliquot should be concentrated at least 100-fold prior to electrophoresis and immunofixation. Dipstick and sulfosalicylic acid techniques are not sensitive enough to detect small quantities of monoclonal free light chains and should not be used as screening tests for this purpose.

Atrophy of Villi With Hypertrophy and Hyperplasia of Paneth Cells in Isolated (Thiry-Vella) Ileal Loops in Rabbits: Light-microscopic studies

Thiry-Vella loops in rabbit ileum were prepared by a new technique and were studied 18 hr to 49 days postisolation. The loops became grossly shortened after 14 days. Histologically, some shortening and blunting of villi was detectable as early as 4 days postisolation, and with prolonged isolation the changes became marked. Reduction in epithelial cell height and in brush border thickness were noted, and goblet cells were increased somewhat in size and prominence. Yet there was only slightly increased chronic inflammation in the mucosa and acute inflammation was uncommon, suggesting that mucosal injury was minimal. Furthermore, mean epithelial mitotic indices for the crypts did not rise and were generally reduced. Striking hyperplasia and hypertrophy of Paneth cells associated with mitotic figures in Paneth cells accompanied the atrophic changes in the villi. Reimplantation of loops into the bowel 3 weeks after isolation led to complete reversal of all changes, including hyperplasia of Paneth cells. On the other hand, regular perfusion of loops with a solution containing a large variety of nutrient substances failed to reverse the mucosal changes. It was concluded that atrophy of villi in isolated ileum of the rabbit occurred mainly because one or more substances contained in the chyme are needed to maintain normal mucosal architecture. These substances probably help regulate epithelial cell turnover and may well be endogenous in origin. Loss of substances in the chyme after loop isolation may also have led to Paneth cell hyperplasia. Alternatively, the Paneth cell changes and atrophy of villi might have been related in a cause and effect way.

Serum free light chain (FLC) measurement can aid capillary zone electrophoresis in detecting subtle FLC-producing M proteins.

We hypothesized that using a free light chain (FLC) assay as an adjunct to capillary zone electrophoresis (CZE) could improve detection of lymphoplasmacytic processes. We prospectively studied 1,003 consecutive serum samples submitted for routine protein electrophoresis and/or immunofixation electrophoresis by CZE and FLC. Samples from patients previously characterized as having M proteins were excluded. Protein electrophoresis was read by a pathologist unaware of the FLC results. Sixteen cases revealed an abnormal free kappa/lambda ratio in which CZE did not demonstrate an M protein. Nine cases of B-lymphocyte or plasma cell proliferative processes were detected by an abnormal free kappa/lambda ratio in which CZE did not demonstrate an M protein. Cases with low free kappa/lambda ratios included 1 chronic lymphocytic leukemia (CLL), 1 IgM lambda with aplastic anemia, and 1 lambda light chain myeloma. Cases with high free kappa/lambda ratios included 2 CLL, 1 lymphocytosis (possibly early CLL), 1 kappa light chain myeloma, 1 atypical lymphoma with neuropathy, and 1 nonsecretory myeloma. Addition of the free kappa/lambda ratio to CZE increases the yield of lymphocyte and plasma cell proliferative processes detected by 56%.

Antinuclear antibody testing

The ANA test is an excellent screening test for patients with SLE and a few other connective tissue diseases. The LE cell preparation is an assay that is subjective and costly. Because of the presence of a superior screening test (the ANA) and superior specific auto-antibody tests, the author recommends that the use of LE cell preparations be discontinued. ANA screening tests may be performed either by indirect microscopic serology (usually IFA) or EIA. The latter technique is readily automated and many new products for this screening test have appeared in the past decade. The products differ, however, and laboratories are cautioned to test each in the context of the clinical needs of their clinicians. Proper use of the ANA test requires each laboratory to determine the cutoff used under their conditions of assay. Although either ANA screening test has a high negative predictive value in numerous studies, proper selection of patients to be tested is key to improving the predictive value of a positive result. The American College of Rheumatism criteria are reviewed and recommended as part of the patient selection process for this testing.

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 Peyers 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 Peyers patches and isolated folicles 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 Peyers 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 Peyers 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.

Demonstration by in situ hybridization of the zonal modulation of rat liver cytochrome P-450b and P-450e gene expression after phenobarbital

The various physiological processes that constitute liver function are compartmentalized within the hepatic acinus. The molecular mechanisms modulating the development and maintenance of this hepatocyte heterogeneity have not been defined. The objective of this study was to determine whether transcriptional or posttranscriptional zonal modulation of cytochromes P-450b,e gene expression was responsible for the heterogeneous induction of the P-450 proteins, which is observed after phenobarbital (PB) administration. The exact localization in liver tissue of hepatocytes responding to PB with induction of either P-450b,e mRNA or proteins was established by in situ hybridization and by immunofluorescence, respectively. As demonstrated by quantitative assessment of autoradiographs of approximately 20 hepatocytes located between a terminal portal venule and a hepatic venule, PB induced the P-450b,e mRNA up to sixfold in the 12-15 hepatocytes located closer to the hepatic venules (zones 2 and 3). In contrast, there was only a twofold induction in the 4-6 hepatocytes surrounding the terminal portal venules (zone 1). Quantitative immunofluorescence using an MAb showed that the acinar distribution of PB-induced P-450b,e proteins was similar to that of the mRNA. This combined approach indicated that, most likely, an increased rate of transcription of cytochromes P-450b,e genes in hepatocytes of zones 2 and 3 concomitantly, with a relative lack of activation, or repression, of these genes in hepatocytes of zone 1, were responsible for the heterogeneous phenotype observed after PB administration. Therefore, modulation of gene expression among hepatocytes of the liver acinus is one mechanism by which the functional heterogeneity of hepatocytes is attained. Experiments in which the induction of cytochromes P-450b,e genes was studied after administration of either PB or para-hydroxyphenobarbital, a main hepatic metabolite of PB, suggested that the species involved in the inductive process is the parent PB molecule rather than para-hydroxyphenobarbital.

WHIPPLE'S DISEASE: A REVIEW EMPHASIZING IMMUNOLOGY AND MICROBIOLOGY

Whipples disease is an important and fascinating problem of local immunity in the gastrointestinal tract. Does the disease occur when an organism that is rare in nature infects an individual or do patients with Whipples disease have a definable defect in their immune response that permits infection by a more common agent? Data in recent years indicate that there is only one type of microorganism that causes Whipples disease. It is rod-shaped by electron microscopy and has a definable antigenic pattern by immunofluorescence. Paradoxically (considering its geometry), it reacts most strongly with antisera directed to streptococcal antigens. Patients with Whipples disease do not have a disorder of immunoglobulin synthesis and do not have immune complexes present in their gut walls. Further, although earlier studies indicated that a defective response of T-lymphocytes to PHA is consistently present in these patients even years after therapy, more recent studies have found no consistent defect in mitogenic responses to PHA, CON A, or PWM. Also, recent studies indicate that mononuclear cells from patients with Whipples disease usually mediate antibody-dependent cell-mediated cytotoxicity as well as controls, although spontaneous cell-mediated killing may be decreased. All these studies suggest that the defect is not primarily of lymphocytes, rather it is more likely that a defect exists in monocytes and macrophages. Future studies on Whipples disease should add to existing knowledge of how the immune processes intracellular microorganisms.

Barrett's esophagus complicating achalasia after esophagomyotomy: a clinical, radiologic, and pathologic study of 70 patients with achalasia and related motor disorders

Of 70 patients with achalasia and related motor disorders, 3 developed Barretts esophagus 5, 8, and 15 years after esophagomyotomy. One of the three had dysplastic changes in the Barretts mucosa. Although an increased incidence of gastroesophageal reflux, esophagitis, and stricture are well-known complications after esophagomyotomy, the development of Barretts mucosa has been only recently recognized. Diagnosis of Barretts esophagus in such patients is difficult and requires a high index of awareness by the radiologist and an endoscopic biopsy for definitive diagnosis. The cumulative effects of achalasia and Barretts esophagus predispose these patients to higher risks of developing esophageal carcinoma.