The human thymus during aging.

The human thymus is required for establishment of a normal T cell repertoire in fetal development, as children born without a thymus (DiGeorge Syndrome) lack thymus-derived (T) and T cell immunity. While the function of the thymus in children for production of new T cells is clear, it has not been obvious that the adult thymus can produce significant numbers of new T cells. Until recently, no assays were available to directly evaluate postnatal thymic function. This paper reviews work on human thymic aging at Duke University School of Medicine and discusses the relevance of this work to devising new strategies for T cell immune reconstitution in man.

The localization of hyaluronan in normal and rejected human kidneys.

Hyaluronan (HYA) is a large glycosaminoglycan with a high capacity to immobilize water. Increased levels of HYA have previously been observed in plasma as well as in affected tissues in various inflammatory conditions. The morphological localization of HYA has, however, not been described in normal or rejected human kidneys. Using a recently developed method for localization of HYA in tissue sections by means of a biotin-labeled hyaluronan binding protein used as a probe, we have now investigated the distribution of HYA in normal and irreversibly rejected human kidneys. In the normal kidney HYA was essentially confined to the medulla. In the rejected kidneys increased amounts of HYA were observed primarily in the cortex and in sclerotic vessels. Incubating tissue sections with hyaluronidase abolished the staining for HYA, showing the specificity of the staining procedure. The increased amounts of HYA of the rejected kidney may play a role in local edema formation, and thereby alter graft function.

Localization of hyaluronan in regions of the human female reproductive tract.

Accumulation of hyaluronan has previously been observed in various organs as an inflammatory response. To study the presumed connection between infertility due to a tubal factor and inflammation, we performed an analysis of the hyaluronan distribution in biopsy specimens from the female reproductive tract, using a biotinylated hyaluronan binding protein (HABP) as a histochemical probe. In normal specimens hyaluronan was localized in the dense, irregular connective tissue surrounding blood vessels of various sizes. Smooth muscle and columnar epithelium were devoid of hyaluronan. The isthmic part of the normal Fallopian tube showed moderately intense staining of the entire lamina propria, whereas normal fimbriae stained weakly. No cyclic changes in hyaluronan content were observed. In biopsy specimens from women with infertility due to a tubal factor, intense staining, stronger than in normal tubes, was detected in the adhesions and in the lamina propria of sactosalpinx. This may indicate that infertility due to a tubal factor is associated with an ongoing inflammatory and/or proliferative process.

Localization of hyaluronan in normal breast tissue, radial scar, and tubular breast carcinoma.

Hyaluronan (hyaluronic acid [HYA]) is one of the extracellular matrix components involved in normal cell physiology and is localized mainly in bodily fluids and connective tissues. Increased amounts of HYA in serum have been demonstrated in a number of neoplastic and inflammatory conditions, among them breast cancer. Tubular breast carcinoma (TC) and radial scar (RS) are two breast lesions that microscopically display characteristic stromal alterations and possess gross and microscopic similarities. Due to the importance of HYA as a component of the extracellular matrix, we investigated its presence in these lesions and in normal breast tissue. Using a biotinylated HYA-binding region for the in situ detection of HYA, we noted an increased amount of HYA in both TC and RS as compared with that in normal breast tissue specimens. A strong reactivity was observed predominantly around glandular structures and in the interlobular stroma of both TC and RS. Perivascular HYA staining also was distinctly observed in these lesions (TC and RS). Some HYA was observed in the connective tissue of the intralobular regions, around small blood vessels, and in the perivascular connective tissue of the normal breast. The distribution of HYA adjacent to the epithelium in the normal breast suggests a role for HYA in the interaction between epithelium and stroma of the normal breast. Its increase in the connective tissue of both TC and RS reflects the derangement of the stroma commonly observed in these conditions and supports the notion that these lesions may be associated.

Expression of an interferon-gamma-like substance in normal and transplanted rat heart tissue.

The presence of interferon-gamma-like molecules has been reported not only in lymphocytes, but also in certain nerve cells and in normal skeletal muscle. We have studied the reactivity of the anti-interferon-gamma monoclonal antibody DB1 with frozen sections of normal and transplanted rat hearts. Cardiac grafts from PVG donor rats were transplanted to syngeneic PVG recipients or allogeneic Wistar/Kyoto recipient rats with the use of an accessory cervical heart transplantation technique. The allogeneic heart transplants were harvested 4 days and the syngeneic grafts 4 weeks after transplantation. In normal hearts there was a weak but distinct reactivity with the anti-interferon-gamma antibody in most muscle cells. In addition, some lymphocytes and the Purkinje fibers were positive. Hearts transplanted over an allogeneic barrier revealed that staining for interferon-gamma on muscle cells was substantially increased whereas no or only a moderate increase in the anti-interferon-gamma staining was seen in hearts transplanted to syngeneic recipients. These data indicate that interferon-gamma present in rat myocyte may be involved in the pathophysiology of graft rejection and also suggest that interferon-gamma may be of importance for the function of normal rat heart muscle cells.

Expression of interleukin-2 receptor alpha (IL-2R alpha) mRNA and protein in advanced epithelial ovarian cancer.

Advanced epithelial ovarian cancer has recently been identified by us to be associated with elevated serum and ascitic levels of the soluble Interleukin-2 receptor alpha (sIL-2R alpha). To determine the cellular source of sIL-2R alpha, the expression of IL-2R alpha was assessed at the mRNA and protein level in peripheral blood mononuclear cells (PBMC), in ovarian cancer ascitic cell infiltrates and in primary and metastatic epithelial ovarian cancer lesions by immunochemistry, by flow cytometric analysis and by in situ hybridization (ISH). Normal PBMC and the PBMC from ovarian cancer patients had a low or undetectable level of IL-2R alpha mRNA and of IL-2R alpha cell-surface protein expression. Flow cytometric analysis of the heterogeneous ascitic infiltrates revealed few cells positively expressing cell-surface IL-2R alpha. By immunocytochemistry, 1-2% of leukocytes in the ascitic infiltrates were IL-2R alpha+. Cytologically these IL-2R alpha+ cells were lymphocytes. Frozen sections of primary and metastatic ovarian cancer lesions showed sparse lymphocytic infiltration and very small numbers of these tumour infiltrating lymphocytes (TIL) were IL-2R alpha+. In situ hybridization demonstrated that although less than 2% of leukocytes in the ascitic infiltrate had detectable levels of IL-2R alpha mRNA, there was a wide range in the level of mRNA expression in these positive cells. The cells expressing IL-2R alpha mRNA had the cytologic characteristics of lymphocytes. Similarly, in the frozen sections of the solid tumours, there was a range in the level of IL-2R alpha mRNA expression in the few TIL that expressed IL-2R alpha. Importantly, ovarian cancer cells and mesothelial cells did not express IL-2R alpha mRNA or IL-2R alpha protein. Our observations lead us to conclude that lymphocytes are the main, if not the only, source of sIL-2R alpha in ovarian cancer patients. Although cells expressing IL-2R alpha were relatively few in number, as the source of the high levels of sIL-2R alpha, they may contribute to the immunosuppression of ascitic lymphocytes in advanced epithelial ovarian cancer.

Histochemical localization of hyaluronan in psoriasis, allergic contact dermatitis and normal skin.

In suction blister fluid from active psoriatic lesions we have previously found elevated concentrations of hyaluronan. The aim of this investigation was to study the localization of hyaluronan with a histochemical method, in biopsy specimens from lesions of 13 patients with progressive psoriasis. Ten normal subjects and seven patients with allergic contact dermatitis were also studied. In normal epidermis the highest intensity of hyaluronan staining was found in the intercellular spaces in the middle and upper spinous layer, whereas the staining was much weaker in the basal layer. No hyaluronan was detected in the granular layer or in the orthokeratotic stratum corneum. In the dermis there was pronounced staining of the papillary dermis and around the sebaceous glands, sweat glands, hair follicles and blood vessels. In six of the 16 specimens from psoriatic lesions the normal epidermal meshwork of hyaluronan was partly absent and replaced by diffuse staining of both the spinous and the basal layer. In the remaining ten of these 16 specimens the same type of meshwork was found in stratum spinosum as in normal skin. The parakeratotic stratum corneum contained hyaluronan, in contrast to the normal stratum corneum, where no hyaluronan was present. The pattern of hyaluronan staining in the dermis of the psoriatic lesions did not differ from that in normal dermis. In the majority of the allergic patch test reactions the junction was less distinct than in normal skin between dermis and epidermis and the normal hyaluronan pattern of the basal layer was abolished and replaced by a diffuse staining throughout the layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid fluorescein and protein assay method for fluorescent-antibody conjugates.

A method is presented for the rapid determination of the fluorescein content, protein content, and fluorescein-to-protein ratio for immune globulin conjugates with fluorescein isothiocyanate as the fluor. This method is based on the absorbance of the fluorescent antibody at those wavelengths primarily associated with the fluorescein and gamma-globulin fractions, and permits these materials to be determined by a single nondestructive analytical procedure. A small sample of the fluorescent antibody, in many cases 0.1 ml or less, is adequate for the above determinations. A nomograph is presented which allows simultaneous determination of the materials from the observed absorbance at the two wavelengths. The method is sufficiently accurate for most applications of the fluorescent-antibody techniques. Although this procedure has been developed primarily for fluorescent-antibody conjugates prepared from rabbit gamma-globulin, it can be used directly for antibodies prepared from other animals provided the gamma-globulin is relatively free from albumin.

Systemic injection of group A streptococcal peptidoglycan-polysaccharide complexes elicits persistent neutrophilia and monocytosis associated with polyarthritis in rats.

The perpetuation of inflammatory changes within joints elicited by persisting, poorly biodegradable group A streptococcal cell walls (peptidoglycan-polysaccharide complexes [PG-PS]) is well documented. Chronic changes in the bloodstream induced by PG-PS have not been described previously. We demonstrated that leukocytosis occurs within 3 days after intraperitoneal injection of PG-PS and remains elevated 20 weeks later. Chronic neutrophilia, monocytosis, and lymphocytosis were observed in all experiments. Chronic changes in platelet, erythrocyte, and reticulocyte counts were not seen. The newly documented leukocytosis, lasting for months after PG-PS administration, provided a circulating pool of leukocytes that may participate in chronic inflammatory events in the joint. Although the central role of the macrophage in PG-PS-mediated inflammation has been emphasized (F. G. Dalldorf, W. J. Cromartie, S. K. Anderle, R. L. Clark, and J. H. Schwab, Am. J. Pathol. 100:383-402, 1980), the polymorphonuclear cell may be involved in periods of exacerbation of streptococcal cell wall-mediated polyarthritis. This was supported by our observations that neutrophilia and monocytosis correlate well with the degree of chronic joint inflammation.

Local increase in hyaluronic acid and interleukin-2 in the capsules surrounding silicone breast implants.

Connective tissue disease-like illness has been associated with silicone breast implants. However, no data are currently available on the immunopathology of the capsule surrounding the breast implants. Sera from women with breast implants were collected and assayed for interleukin-6 (IL-6), IL-2, and hyaluronic acid. Capsular biopsies were stained with a probe for HYA or with monoclonal antibodies specific for human macrophages (CD68), T cells (CD4), IL-6, and IL-2. Control specimens consisted of breast biopsies from women undergoing reduction mammoplasty. Our results revealed an increased local amount of hyaluronic acid in the capsule of patients with breast implants compared with control breast tissue. The HYA was localized extracellularly in areas containing fibrosis and cellular infiltrates. The infiltrating cells were determined to be primarily macrophages and T cells. No IL-6 was localized in any of the tissue sections. In contrast, large amounts of IL-2 were found in regions of infiltrating lymphocytes. No significant increase in IL-6, IL-2, or hyaluronic acid was found in the sera. The role of hyaluronic acid and cytokines in the inflammatory response in the capsules of silicone breast implants is discussed.

Correlation between increased hyaluronan localized in arthritic synovium and the presence of proliferating cells. A role for macrophage-derived factors.

OBJECTIVE: To determine whether the increased levels of circulating hyaluronan seen in patients with arthritis also occur locally. METHODS: Biopsy specimens of normal synovium and synovium from patients with various arthropathies were studied using histochemical and immunohistochemical staining procedures, to determine the tissue distribution of hyaluronan and infiltrating cells. RESULTS: Hyaluronan was found in increased concentrations in inflamed tissues, and was co-localized in sites containing Ki-67+ cells. In vitro analyses showed that macrophage-released factors increased hyaluronan production by fibroblasts. Hydrocortisone inhibited this in vitro production of hyaluronan. CONCLUSION: Edema and swelling seen in inflamed joints may be due to the presence of large amounts of hyaluronan. One possible mechanism of action of corticosteroids in the alleviation of acute joint inflammation may occur via the inhibition of hyaluronan production.

Localization of hyaluronan in various muscular tissues. A morphological study in the rat.

The histochemical distribution of hyaluronan (hyaluronic acid, HYA) was analysed in various types of muscles in the rat by use of a hyaluronan-binding protein (HABP) and the avidin-biotin/peroxidase complex staining procedure. Microwave-aided fixation was used to retain the extracellular location of the glycosaminoglycan. In skeletal muscles, HYA was detected in the connective tissue sheath surrounding the muscles (epimysium), in the septa subdividing the muscle fibre bundles (perimysium) and in the connective tissue surrounding each muscle fibre (endomysium). HYA was heterogeneously distributed in all striated muscles. In skeletal muscles with small fibre dimensions (e.g., the lateral rectus muscle of the eye and the middle ear muscles), HYA was predominantly accumulated around the individual muscle fibres. Perivascular and perineural connective tissue formations were distinctly HYA-positive. In cardiac muscles, HYA was randomly distributed around the branching and interconnecting muscle fibres. In comparison, smooth muscle tissue was devoid of HYA.

Cytokine gene expression: analysis using northern blotting, polymerase chain reaction and in situ hybridization.

We describe here the use of northern blotting, PCR and in situ hybridization for the analysis of cytokine gene expression. These techniques, each with their advantages and disadvantages, have been used to monitor cytokine gene expression in sites of immune reactivity and in the developing thymus. Whilst expression of a gene usually correlates well with protein production from that gene, this may not always be the case. The development of methods to analyze protein production in situ, for instance by immunohistochemistry, together with analysis of mRNA expression will allow us to begin to understand the role of cytokines within the immune system of the intact animal.

Lung hyaluronan during development: a quantitative and morphological study.

Hyaluronan, an important constituent of developmental interstitium in fetal lungs, mediates cell-to-cell interactions and thereby directs migrating cells. Furthermore, because of the polyionic nature of the molecule, hyaluronan forms open, hydrated matrices that provide channels for migrating cells. This hydrated matrix undergoes contraction before birth. However, continued growth of the lung in the perinatal period requires newly synthesized hyaluronan. This study's purpose was to elucidate the changes in lung hyaluronan concentration and distribution in the perinatal period. We studied rabbits at days -6, -4, -2, -1, 0, +2, and +4 with respect to term, as well as adult rabbits. We found that hyaluronan concentration was highest in the youngest fetuses studied [682 +/- 115 micrograms/g dry wt (means +/- SD)]. However, hyaluronan concentration decreased to 129 +/- 12 micrograms/g dry wt just before birth then returned to 366 +/- 111 micrograms/g dry wt at day +4; these values were similar to adult values. We found hyaluronan staining decreased during late gestation, particularly in the interalveolar region. In the postnatal period, hyaluronan staining increased in the visceral pleura and, to a lesser extent, beneath the epithelium of the bronchioles. Hyaluronan did not reaccumulate in the interalveolar region in the postnatal period. Our data suggest a change in the predominant role of lung hyaluronan during the perinatal period. Before term, hyaluronan facilitates morphogenesis. However, hyaluronan's major role in neonatal lung is most likely in regulation of fluid balance in interstitium.

Localization and quantity of hyaluronan in urogenital organs of male and female rats.

The histochemical distribution of hyaluronan was analysed in various urogenital organs of male and female (non-pregnant and pregnant) rats by use of a hyaluronan-binding protein and avidin biotin/peroxidase staining. Microwave-aided fixation was used to preserve the extracellular location of hyaluronan. The concentrations of hyaluronan in the different tissues were measured with a highly sensitive radio-assay. Hyaluronan accumulated predominantly in the connective tissue around smooth muscle fibres and in the subepithelial lamina propria. Abundant hyaluronan also occurred in perivascular and perineural connective tissue. In the female urogenital organs, hyaluronan content was high in the vagina and urinary bladder, and highest in the vagina during pregnancy. In the uterus, the surface epithelium of the endometrium stained intensely. In the ovary, the zona pellucida of the oocyte and the theca interna cell layer of the follicles and the follicular fluid of mature follicles exhibited prominent staining. The corpus luteum was devoid of hyaluronan, whereas enlarged corpora lutea of pregnancy exhibited weak, patchy staining. In male urogenital organs, staining for hyaluronan was absent from the testis and epididymis, whereas the erectile connective tissue of the penis stained intensely. The hyaluronan concentrations were high in penile tissue and urinary bladder, while testis, epididymis and the ductus deferens contained only little hyaluronan.

Borrelia burgdorferi decreases hyaluronan synthesis but increases IL-6 production by fibroblasts.

Despite the prevalence of clinical data on human Lyme disease, little is known about the immunopathologic effects of the causative organism on the host. We studied the effect of Borrelia burgdorferi on hyaluronan (hyaluronic acid, HYA) production and the effect on interleukin-6 (IL-6) synthesis by cultured fibroblasts. The cell line employed in this study produced an average of 1406 ng of hyaluronan/ml within 48 h. Using both a morphological staining protocol and a quantitative radiometric assay, we noted that in the presence of a low dose of Borrelia (9.4 x 10(5) cells/ml) the hyaluronan production decreased to an average of 1008 ng/ml, a significant difference (p < 0.05) from the amount of hyaluronan produced by the cells alone. The reduction was even more significant (p < 0.01) when a higher dose of Borrelia (9.4 x 10(6) cells/ml) was used giving an average hyaluronan concentration of 682 ng/ml. In contrast, we found that Borrelia stimulated the cells to produce IL-6 from a baseline of 293 pg/ml to a maximal value of 842 pg/ml (p < 0.01). The spirochetes had no significant effect on cell viability, nor were we able to demonstrate invasion of the cells by the bacteria. Both a decrease in hyaluronan and an increase in IL-6 may correlate with the pathogenicity of Lyme disease in man.