Immunohistochemical localization of three different immunoglobulin classes in the Harderian gland of young chickens

The chicken Harderian gland (HG) was investigated using single immunohistochemical staining for one of the three different immunoglobulins (Igs) followed by Alcian blue/periodic acid Schiff (AB/PAS) staining and triple immunohistochemical staining for all of the Igs with hot water treatment. In the HG of 5-week-old chickens, IgG-containing plasma cells were more frequent than IgA- and IgM-containing cells. These numerous IgG-containing cells were predominantly accumulated in the central region of the stroma, whereas a small number of IgA- and IgM-containing cells were scattered in the peripheral region of the stroma. Also, the plasma cells containing PAS-positive Russell bodies (RBs) exhibited distinct immunoreactivity for one of the Igs, being inversely proportional to the intensity of PAS reaction. The RB-containing cells positive for IgA were more frequent than those positive for IgM, whereas those positive for IgG were very rare. The most distinct feature of the IgG-containing plasma cells was a PAS-positive globule located close to the nucleus. Triple immunostaining with hot water treatment simultaneously identified these three Igs in normal plasma cells and RB-containing ones in the stroma of the chicken HG.     

Reproducibility of counting immunoglobulin-containing cells in colonic mucosal biopsies

Counting immunoglobulin (Ig)-containing cells in colonic mucosal biopsies can help to objectively support the differential diagnosis of ulcerative colitis and Crohn's disease. Before a method for counting Ig-containing cells can be applied in a clinical setting, however, its reproducibility must be determined. This study investigated the reproducibility of two different methods for counting such cells. The use of a light microscope with an ocular grid resulted in a slightly better reproducibility than did the use of a projection microscope with a graphics tablet. Moreover, the ocular grid method had a higher efficiency. The counting of IgM- and IgG-containing cells had a considerably higher reproducibility than did the counting of IgA-containing cells. To determine the minimal number of cells to be counted in order to ascertain a stabilized mean number of Ig-containing cells, the running means of counts of Ig-containing cells were calculated for two observers. When at least 600 Ig-containing cells (i.e., two to four fields) were counted, the interobserver variation of the running means was less than 10% for IgA and IgG counts and less than 5% for IgM counts. Since earlier studies showed differences in the counts of IgA-, IgG- and IgM-containing cells between ulcerative colitis and Crohn's disease to be, respectively, 3%, 25% and 28%, the results of the present study suggest that the proposed counting method can be useful in the differentiation between these entities.     

Influence of fixation and immunohistological technique on accuracy, precision and inter-observer reproducibility of plasma cell counting.

Recently two highly sensitive and specific diagnostic criteria for Sj?gren's syndrome based on percentages of IgA-, IgG-, and IgM-containing plasma cells measured in immunohistologically stained labial salivary gland tissue have been described. The reliability of such a criterion is dependent on the accuracy, precision and inter-observer reproducibility in plasma cell counting. The present study evaluates the effect of tissue fixation and immunohistological procedures on the aforementioned factors. Immunoglobulin (Ig)-containing plasma cells in sections of lamellated submandibular salivary gland tissue, alternately fixed in a 4% buffered formol solution or formol-sublimate solution and stained with an indirect immunoperoxidase and unlabelled peroxidase anti-peroxidase (PAP) method respectively, were enumerated by three independent observers. Relative numbers of Ig-containing plasma cells appeared to be less sensitive for systematic errors due to tissue fixation and immunohistological procedure than absolute numbers of Ig-containing plasma cells. The best inter-observer reproducibility of plasma cell counts was obtained in sections from formol sublimate-fixed specimens stained according to the PAP procedure.     

Distribution of T-cell subsets and immunoglobulin-containing cells in nasal-associated lymphoid tissue (NALT) of chickens

The present study demonstrated the localization of the T-cell subsets (CD4+ and CD8+) and immunoglobulin (Ig)-containing cells (IgA, IgM, and IgG) in the nasal mucosa and its accessory structures. These lymphoid structures may be compared with nasal-associated lymphoid tissue (NALT) of rats and mice. In the chicken NALT, T-cell subsets were more widely distributed than Ig-containing cells, especially in large lymphoid accumulations restricted to the respiratory mucosa in the nasal cavity and the nasolacrimal duct. These lymphoid accumulations in the mucosa of the nasal cavity and nasolacrimal duct consisted of widely distributed CD8+ cells and deeply aggregated CD4+ cells adjacent to large germinal centers. In these lymphoid accumulations, IgG-containing cells were more frequently observed than IgM- and IgA-containing cells. T-cell subsets, predominantly CD8+ cells were more widely distributed in the duct epithelium of the lateral nasal glands than Ig-containing cells. Moreover, numerous CD8+ cells and a few Ig-containing cells were found in the chicken salivary glands, especially around the orifice of their ducts into the oral cavity. Therefore, it seems likely that the chicken NALT plays an important part in the upper respiratory tract, with a close relationship to the paraocular immune system.     

B cell maturation in the chicken Harderian gland

We have characterized maturation of B lymphocytes in the chicken Harderian gland. Expression of Ig genes was studied by using lambda L and mu H chain-specific DNA probes. In unstimulated chickens, the concentration of mu H chain and lambda L chain mRNA in the Harderian gland was observed to be greater than 8 times higher than in the bursa of Fabricius or spleen. By using in situ hybridization, the plasma cells expressing mu mRNA were located in central area of the gland packed around the tubules. Antibodies produced by the Harderian plasma cells were measured from the tears before and after antigenic stimulation. In unstimulated chickens high levels of total IgM, IgA, and IgG were observed. After ocular stimulation with tetanus toxoid, specific antitetanus IgG and IgA antibodies appeared in the tears but IgM antibodies were barely detectable. These results indicate that after antigenic stimulation the Harderian B cells rapidly mature through IgM secretion to the production of IgG or IgA. Southern blot analysis of the Harderian total genomic DNA showed strong rearrangement in the lambda L chain locus. In contrast, the band indicating major rearrangement in the mu H chain locus gave a very poor hybridization signal, indicating deletion of C mu genes in the Harderian gland DNA. As a conclusion, our present data indicate for the Harderian gland a role in terminal B cell differentiation and Ig class switch.     

The effects of adrenalectomy on the harderian gland of the Wistar albino rats

Harderian glands of the Wistar albino rats normal and adrenalectomized were investigated by light microscopy. In normal, these glands have a tubuloalveolar structure. The gland is located in the medio posterior aspect of the orbit. It is lobulated and appears homogeneous in colour and texture. Harderian gland consist of tubules with wide lumina lined by a single layer of columnar epithelial cells surrounded by myoepithelial cells within their basal lamina. It contains porphyrin pigment which is stored as solid intraluminal deposits. The glandular epithelium possesses two cell types, termed A and B. Type A cells are more numerous. The single excretory duct of the gland is directly continuous with endpieces at the hilus and opens nasally and ventrally to the third eyelid. The excretory duct is accompanied by many acini of small serous glands around it. The tubuloalveoli of the gland is not divided into lobules. There is no branched duct system within the gland. The secretion seems to be associated with porphyrins, is essentially released by exocytosis, but holocrine secretion also occurs. The single excretory duct is lined by a stratified epithelium. The gland is surrounded by a collagenous capsule. The adrenalectomy, caused degenerative changes in the glands. Epithelial height was lower than in normal gland epithelium. Most of the acini were completely disorganised. The acinar lumina were filled with porphyrin debris. The results suggest that rat harderian glands are sensitive to adrenal androgen changes in both male and female rats.     

Sexual differences and effects of castration on secretory mode and intracellular calcium ion dynamics of golden hamster Harderian gland

The aim of the present work was to study the sexual differences in secretory mechanisms and intracellular calcium ion dynamics in the Harderian gland of the golden hamster. In both sexes the Harderian gland consisted of small and large lobes. In the intact control male glands the secretory portions of both lobes showed wide lumina that contained secretory material and cytoplasmic fragments, suggestive of the occurrence of exocytosis and apocrine secretion. After perfusion with HEPES-buffered Ringer's solution containing 10 microM carbamylcholine (CCh), the glandular cells showed features of enhanced secretion and a rise in intracellular calcium concentration ([Ca2+]i). In the intact control female gland the lumina of most secretory portions in the large lobe contained porphyrin accretions, and exocytosis was the sole secretory mechanism. Stimulation of the large lobe with 10 microM CCh did not raise [Ca2+]i or cause enhanced secretion. The small lobe in females resembled the male gland in secretory functions, and CCh administration caused enhanced secretion and a rise in [Ca2+]i. Castration in males abolished apocrine secretion; exocytosis became the sole secretory mechanism, and stimulation of the glandular cells with CCh did not cause enhanced secretion or induce a rise in [Ca2+]i. To the contrary, in females, castration restored apocrine secretion and CCh administration caused enhanced secretion and a rise in [Ca2+]i. Castration did not affect the secretory mechanisms and the effect of CCh on the glandular cells in the small lobes of both male and female glands. The present study points to the possibility that sex hormones may control the functioning or expression of muscarinic receptors in the Harderian gland of the golden hamster.     

Bursectomy of chicken embryos at 60 hours of incubation leads to an oligoclonal B cell compartment and restricted Ig diversity

Chickens that have been surgically bursectomized at 60 h of embryonic development usually generate Ig producing B cells; however, the bursectomized chickens are incapable of specific antibody responses, even after repeated immunization. In the present work, we analyzed the molecular basis of this immunodeficiency. In the bursectomized chickens, DNA sequencing revealed a repertoire of Ig L and H chains with a low number of different V-J and V-D-J joints, indicating an oligoclonal B cell compartment. In addition, the L and H chains belonging to each B cell clone had similar gene conversion events in the V region. In situ hybridization to Harderian gland tissue sections showed, that B cells of the bursectomized chickens were, however, capable of terminal plasma cell maturation. Thus, in chickens that were lacking the bursal microenvironment, 1) only a few B cell precursors differentiated into mature Ig-producing B cells, 2) low rate of gene conversion resulted in restricted Ig diversity. Regarding the chicken B cell differentiation, the present data support a model that the induction of B cell differentiation is a bursa-independent event, whereas the bursa of Fabricius has a crucial role in the amplification and diversification of the embryonic B cell repertoire.     

The Harderian gland of the Mexican volcano mouse Neotomodon alstoni alstoni (Merriam 1898): a morphological and biochemical approach

The Harderian glands of rodents are large intraorbital exocrine glands with histologic organization that varies among mammalian species. Here we describe some ultrastructural and biochemical features of the Harderian gland in the Mexican volcano mouse Neotomodon alstoni alstoni, a species of restricted habitat. The Harderian glands from male and female adult mice were dissected, processed and embedded in Epon 812 for light and electron microscopy studies. Porphyrin and total lipids were biochemically determined. The macroscopic appearance of the Harderian gland is similar in the male and female. The gland is a bilobulate structure, situated in the orbit towards the posterior side of the eyeball, of whitish color and is surrounded by a connective tissue capsule. The male gland is slightly heavier (127 mg) than that of the female (113 mg). The Harderian gland shows a tubulo-alveolar organization and is composed exclusively of one type of secretory cells. No branched duct system within the gland was found. Adrenergic nerves endings and mast cell were observed in the interstices of the alveoli. Male and female glands produce similar levels of porphyrins. Triglyceride levels were significantly higher (P < 0.05) in the female compared to the male. Abundance of lipids could induce corneal lubrication of the Harderian gland which may confer a protective and adaptative function to the volcano mouse in its natural habitat during the dry and cold seasons.     

Immunoglobulin isotypes in plasma cells of normal and athymic mice.

The distribution of plasma cells was determined in various lymphoid tissues and exocrine glands of athymic (nude) mice. Compared to values for normal mice, the total number of plasma cells in organs of athymic mice showed a variable decrease as follows: 0%, small intestine; 29%, respiratory tree; 33%, spleen; 50%, lymph nodes; 75%, lactating mammary gland; 85%, Peyer's patches; and 90%, parotid gland. Plasma cells containing IgG₁ or IgA showed the greatest decrease, whereas IgM-containing plasma cells were actually increased by 100% or more in most organs. In exocrine glands the absolute deficit of IgA-containing plasma cells was most marked in the parotid and lactating mammary gland, and least in the small intestine. All lymphoid tissues had a striking deficit in the absolute numbers of IgA as well as IgG, plasma cells. Total plasma cell numbers and their isotype distribution were similar for BALB/c +/+ (homozygous) and +/nu (heterozygous) mice.     

Harderian gland dacryoadenitis in rats resulting from orbital bleeding

Localized necrotizing inflammation of the Harderian gland was a sequela to obtaining blood samples from rats by the orbital bleeding technique. The lesion affected a few adjoining lobules that were adjacent to ocular muscles, in contrast to the widespread inflammation of the Harderian gland which results from sialodacryoadenitis virus infection. Lesions resolved in 4 weeks by atrophy of acini in affected lobules with associated mononuclear cell inflammation and fibrosis.     

The Harderian gland of the Cheesman's gerbil (Gerbillus cheesmani ) of the Kuwaiti desert

The Harderian gland is a large orbital structure. Several functions have been ascribed to the gland such as lubrication of the eye, a source of pheromones, thermoregulartory lipids and photoprotective secretions and a part of the retinal-pineal axis. In the present study, the Harderian gland of the Cheesman's gerbil, Gerbillus cheesmani, is described for the first time. The gland is located around the posterior portion of the eyeball. The gland is compound tubular, surrounded by a thin connective tissue capsule. Only one secretory epithelial cell type was recognized, characterized by the presence of lipid vacuoles and cytoplasmic slashes in high numbers; the former being more concentrated towards the apical part while the latter being more concentrated towards the central and basal parts. Some of the cytoplasmic slashes contained electron dense filamentous structures. Similar structures were observed in the lipid vacuoles. Thus, a functional relationship between the cytoplasmic slashes and the lipid vacuoles is suggested. A unique structure was observed, termed dome-like cells, located between the epithelial cells and the basement membrane. These cells were characterized by the extensive presence of pleomorphic mitochondria and compact lamellae of granular endoplasmic reticulum (GER) in the form of finger prints. The gland was found to be actively secreting porphyrins as well as lipids. Cellular debris was also seen in the tubular lumina. Myoepithelial cells with their spindle shape and elongated nuclei were evident between the basement membrane and the secretory epithelium. Sparse interstitial tissue was observed in-between the gland tubules of both male and female gerbils. Macrophages, dendritic melanocytes and lymphocytes are the most represented cellular components of the interstitium. Further studies are required to investigate the function of the dome-like cells as well as the role of lymphocytes in the rodents Harderian gland.     

Sex differences in haem biosynthesis and porphyrin content in the Harderian gland of the golden hamster

Methods are described for the measurement of seven haem biosynthetic enzymes in Harderian gland tissue from male and female golden hamsters. Sex differences were found in five of the seven enzymes. In each case, female tissue exhibited higher activity than male tissue. These differences in enzyme activity are sufficient to account for the major sex difference in porphyrin content in the Harderian gland of this species.     

Effects of endocrine glands and hormone replacement on the mast cell count of the Harderian gland of mice

There are marked sex differences in the Harderian gland of the C3H/He mouse strain, the females having a larger number of mast cells than the males as one of the major differences. Mast cell counts of the Harderian gland were made on male mice subjected to combinations of adrenalectomy, gonadectomy and administration of sex steroid hormones. Castration alone caused a significant increase in the count resulting in about three times the number found in intact males. Castration plus adrenalectomy increased the count over 6-fold, to levels close to those found in female mice. Administration of testosterone or estrone to the mice which had been castrated and adrenalectomized prevented the increase, while progesterone treatment had no effect on the count. Although the number of mast cells in the male Harderian gland was necessarily small compared to either the female gland or that of castrated and adrenalectomized males, no obvious dimorphism could be found microscopically.     

Effect of carbamylcholine on Harderian gland morphology in rats

Summary To determine the effect of cholinergic secretagogue on the Harderian gland of rats, several light- and electron-microscopic parameters were morphometrically assessed at different time intervals after carbamylcholine injection. In controls, two types of glandular cells (type A cells having 40–55 large vacuoles per cell profile and type B cells containing 30–38 smaller vacuoles per cell profile) and myoepithelial cells were recognized. At 5 min after injection of carbamylcholine, when rats secreted bloody tears, many alveoli showing narrower lumina and exocytotic figures in both types of cells were observed. Some vacuoles, which were covered by thin cytoplasmic sheets, protruded into the alveolar lumina. However, there was no evidence of apocrine or holocrine secretion. At 30 min and 120 min after injection, most of the alveolar lumina were dilated, and a pronounced decrease in the number of vacuoles in the glandular cells was observed. At 300 min after injection, the secretory vacuoles in both cell types reaccumulated. Transitional forms between the two cell types were not observed. The two types of Harderian gland cells can therefore be considered independent populations rather than different secretory stages of the same cell. It appears that the secretory process of the Harderian gland of rat is affected by cholinergic stimulation of the two types of glandular cells and of myoepithelial cells.     

Histochemical detection of monoamine oxidase and alcohol dehydrogenase activities in the Syrian hamster Harderian glands: existence of a sexual dimorphism

Summary Monoamine oxidase (MAO) and alcohol dehydrogenase (AD) activities were studied histochemically in the Syrian hamster Harderian gland using tryptamine as substrate and Nitroblue Tetrazolium as the final electron acceptor. No dark: light-related changes were observed. Male type I secretory cells showed an intense MAO reaction. Female type I cells exhibited a moderate MAO activity. Both male and female glands showed a moderate/intense AD-positive reaction. Male type II cells were lacking MAO and AD activities. MAO activity found in the hamster Harderian glands corresponded mainly to MAO type A since treatment with chlorgyline (0.01, 0.1 and 0.5mm) totally inhibited it. The possible role of these two enzymes in Harderian gland indolalkylamine metabolism is discussed.     

Responses of NFR/N inbred mice to very low-dose infections with Ascaris suum

Mice were evaluated for immunological responses and resistance to challenge infections after single infections. When given infections simultaneously with thiabendazole, larval migration to the liver and lungs was blocked but not when mice were given infections only. Enlargement of Peyer's patches was observed after infection followed by increased numbers of IgA-containing cells in the lamina propria of the intestines and increased levels of hepatobiliary IgA. Ig-containing cells were variable in the spleens during the early phase of infection. The observed changes quickly returned to normal control levels and did not change throughout the rest of the experiment. Serum Ig levels did not change dramatically. Challenge with high doses of eggs indicated that mice given single very low-dose infections became highly resistant.     

Sexual dimorphism in the cholinergic innervation of the hamster (Mesocricetus auratus) harderian glands

We study the cholinergic innervation of the Harderian gland in male and female golden hamsters. There is a clear sexual dimorphism in the cholinergic innervation between both sexes. The Harderian gland from male animals contain much more nervous fibers with acetylcholinesterase (AChE) positive reaction than in female. The nervous fibers containing AChE activity are surrounding the acini and blood vessels.     

Gender differences and time course of castration-induced changes in porphyrins, indoles, and proteins in the Harderian glands of the Syrian hamster.

Sexual differences and the effects of orchidectomy were determined for porphyrin and melatonin concentrations and for the activities of the enzymes N-acetyltransferase and hydroxyindole-O-methyltransferase, which synthesize melatonin from serotonin, in the Harderian glands of the Syrian hamster. Porphyrin concentrations in intact males were about 1/400th those of intact females. Castration for 1 week increased male Harderian porphyrin concentrations 10-fold; by 3 weeks, castrated male porphyrin levels were 140 times those of control values. N-Acetyltransferase activity in intact male Harderian glands was about 4 times that of females. Castration led to a drop in N-acetyltransferase activity to female levels within 2 weeks. Hydroxyindole-O-methyltransferase activity was 7 times higher in females than in males and castration had no effect on male Harderian hydroxyindole-O-methyltransferase activity. Neither gender nor castration influenced Harderian melatonin concentrations. Soluble proteins in Harderian glands from male and female hamsters and from male hamsters castrated for 1 and 4 weeks were examined by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The gel profiles revealed several differences among the protein distribution in male and female gland lysates. Orchidectomy led to a female protein pattern within 4 weeks.     

The androgenic effect on the fine structure of the harderian gland in the male hamster

Summary A sexual dimorphism of the hamster Harderian gland at the ultrastructural level has been reported. The effect of testosterone on the fine structure of the gland from castrated male golden hamsters is reported here. Harderian glands from the following three groups of animals were examined at regular intervals up to 60 days after castration: (1) castrated; (2) castratedsham-injected, receiving 0.1 ml sesame oil per day; (3) castrated-testosterone injected, receiving 2mg testosterone propionate in 0.1 ml sesame oil per day. In groups 1 and 2, clusters of cylindrical tubules, typical of the male gland, decreased in number and disappeared almost completely 2 weeks after castration. Membranous structures, typical of the female gland, prevailed in these two groups throughout the remaining period of experiment. On the other hand, these changes were prevented in the group of castrated animals maintained on testosterone propionate. It is concluded that castration modified the ultrastructure of the male hamster Harderian gland toward the female type and that daily administration of testosterone propionate prevented this change.