Critical role of rabphilin-3A in the pathophysiology of experimental lymphocytic neurohypophysitis.

Autoimmune hypophysitis (AH) is thought to be an autoimmune disease characterized by lymphocytic infiltration of the pituitary gland. Among AH pathologies, lymphocytic infundibulo-neurohypophysitis (LINH) involves infiltration of the neurohypophysis and/or the hypothalamic infundibulum, causing central diabetes insipidus resulting from insufficiency of arginine vasopressin secretion. The pathophysiological and pathogenetic mechanisms underlying LINH are largely unknown. Clinically, differentiating LINH from other pituitary diseases accompanied by mass lesions, including tumours, has often been difficult, because of similar clinical manifestations. We recently reported that rabphilin-3A is an autoantigen and that anti-rabphilin-3A antibodies constitute a possible diagnostic marker for LINH. However, the involvement of rabphilin-3A in the pathogenesis of LINH remains to be elucidated. This study was undertaken to explore the role of rabphilin-3A in lymphocytic neurohypophysitis and to investigate the mechanism. We found that immunization of mice with rabphilin-3A led to neurohypophysitis. Lymphocytic infiltration was observed in the neurohypophysis and supraoptic nucleus 1 month after the first immunization. Mice immunized with rabphilin-3A showed an increase in the volume of urine that was hypotonic as compared with control mice. Administration of a cocktail of monoclonal anti-rabphilin-3A antibodies did not induce neurohypophysitis. However, abatacept, which is a chimeric protein that suppresses T-cell activation, decreased the number of T cells specific for rabphilin-3A in peripheral blood mononuclear cells (PBMCs). It ameliorated lymphocytic infiltration of CD3+ T cells in the neurohypophysis of mice that had been immunized with rabphilin-3A. Additionally, there was a linear association between the number of T cells specific for rabphilin-3A in PBMCs and the number of CD3+ T cells infiltrating the neurohypophysis. In conclusion, we suggest that rabphilin-3A is a pathogenic antigen, and that T cells specific for rabphilin-3A are involved in the pathogenesis of neurohypophysitis in mice. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Immunoprofiling of glial tumours of the neurohypophysis suggests a common pituicytic origin of neoplastic cells.

PURPOSE: To analyse the antigen expression profiles of 27 cases of pituicytoma, spindle cell oncocytoma, and granular cell tumour of the sellar region concerning a common pituicytic origin of neoplastic cells. METHODS: Material from 12 female and 15 male patients (13 granular cell tumours of the sellar region, 10 pituicytomas, four spindle cell oncocytomas) collected in the German Registry of Pituitary Tumours between 1993 and 2015 was re-evaluated according to the current WHO classification of tumours of the central nervous system and supplementary immunohistochemistry including S100-protein, CD56, CD68, thyroid transcription factor-1 (TTF-1), and Ki-67 was performed. RESULTS: S100-protein was detected in all 27 tumours and TTF-1 in all 16 tumours that were assessed. Vimentin was expressed in all 13 cases investigated whereas broad spectrum cytokeratin was not detected in any of 14 evaluated cases. GFAP was observed in nine out of 21 cases. 15 out of 17 investigated lesions showed some CD68 expression and five out of 14 cases were labelled with CD56 antibodies. Proliferative activity did not differ significantly between the three tumour subgroups although one primary and one recurrent pituicytoma showed exceptionally high Ki-67-proliferation indices of 15.3 and 12.7 %, respectively (means: granular cell tumour of the sellar region 2.0 %, pituicytoma 2.8 %, spindle cell oncocytoma 2.7 %). CONCLUSIONS: The study confirms and expands earlier data and is in line with the notion that the three tumour types are variants of pituicytoma.

Rabphilin-3A as a Targeted Autoantigen in Lymphocytic Infundibulo-neurohypophysitis.

CONTEXT: Central diabetes insipidus (CDI) can be caused by several diseases, but in about half of the patients the etiological diagnosis remains unknown. Lymphocytic infundibulo-neurohypophysitis (LINH) is an increasingly recognized entity among cases of idiopathic CDI; however, the differential diagnosis from other pituitary diseases including tumors can be difficult because of similar clinical and radiological manifestations. The definite diagnosis of LINH requires invasive pituitary biopsy. OBJECTIVE: The study was designed to identify the autoantigen(s) in LINH and thus develop a diagnostic test based on serum autoantibodies. DESIGN: Rat posterior pituitary lysate was immunoprecipitated with IgGs purified from the sera of patients with LINH or control subjects. The immunoprecipitates were subjected to liquid chromatography-tandem mass spectrometry to screen for pituitary autoantigens of LINH. Subsequently, we made recombinant proteins of candidate autoantigens and analyzed autoantibodies in serum by Western blotting. RESULTS: Rabphilin-3A proved to be the most diagnostically useful autoantigen. Anti-rabphilin-3A antibodies were detected in 22 of the 29 (76%) patients (including 4 of the 4 biopsy-proven samples) with LINH and 2 of 18 (11.1%) patients with biopsy-proven lymphocytic adeno-hypophysitis. In contrast, these antibodies were absent in patients with biopsy-proven sellar/suprasellar masses without lymphocytic hypophysitis (n = 34), including 18 patients with CDI. Rabphilin-3A was expressed in posterior pituitary and hypothalamic vasopressin neurons but not anterior pituitary. CONCLUSIONS: These results suggest that rabphilin-3A is a major autoantigen in LINH. Autoantibodies to rabphilin-3A may serve as a biomarker for the diagnosis of LINH and be useful for the differential diagnosis in patients with CDI.

IgG4-related hypophysitis presenting as a pituitary adenoma with systemic disease.

Hypophysitis is a rare inflammatory disorder that can mimic a pituitary tumor clinically or radiologically. Furthermore, immunoglobulin G4 (IgG4)-related systemic disease is only a just recently characterized disorder. It can manifest as a systemic disease involving multiple organs, including the pancreas, salivary glands, lungs, liver, bile duct, gallbladder, kidneys, and retroperitoneum. It is characterized by a high serum level of IgG4 clinically and dense lymphoplasmacytic infiltration with sclerosis and phlebitis histologically. Herein, we report the case of a man 66 years of age who presented with nausea, vomiting, and poor appetite with a body weight loss of 4 kg. Image study revealed a pituitary infundibulum mass, right-posterior mediastinal and paraspinal masses, as well as infiltrating masses in bilateral kidneys. Therefore, he received a thoracoscopic biopsy for the right-posterior mediastinal and paraspinal masses and a pathologic examination reported an IgG4-related inflammatory pseudotumor. Then, transsphenoidal removal of the infundibulum mass was performed. Histologically, the infundibulum mass represented a IgG4-related hypophysitis manifested as an infiltration of plasma cells, lymphocytes, histiocytes, and some eosinophils with a fair number of IgG4-immunoreactive plasma cells. After the operation was complete, the patient took 5 mg of prednisolone every 2 days for 3 months. A follow-up computed tomography scan revealed improvement of the infiltrating masses in the bilateral kidneys.

Connection between the arcuate nucleus and the posterior lobe of the hypophysis. An angiotensin II immunohistochemical study in rats

The aim of this study was to perform an immunohistochemical study of the, angiotensinergic pathway from the arcuate nucleus (AN) to the posterior lobe of the hypophysis (PLH) of 10-week-old matched normotensive Wistar Kyoto rats (WKY), using our own policlonal antibody raised in mice against Angiotensin II (mouseantiangiotensin II, MAAII). Cells and fibers in the rostroventral and dorsocaudal parts of the AN, the internal zone of the median eminence and PLH showed immunoreactive material for antiangiotensin II. Angiontensin II fibers originating in the anteroventral part of the AN, crossing median eminence (ME) and infundibular stem and arriving at the PLH were also observed (AU)

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Central diabetes insipidus and autoimmunity: relationship between the occurrence of antibodies to arginine vasopressin-secreting cells and clinical, immunological, and radiological features in a large cohort of patients with central diabetes insipidus of known and unknown etiology.

Central diabetes insipidus (CDI) is a rare hypothalamus-pituitary disease due to the deficiency of arginine vasopressin (AVP) synthesis from the hypothalamus and/or secretion from the neurohypophysis. The etiology of CDI is unknown in over one third of cases, classified as idiopathic CDI. The aim of this study was 2-fold: 1) to evaluate the occurrence of circulating autoantibodies to AVP-secreting cells (AVPcAb), and 2) to correlate it to clinical (sex, age of disease onset, disease duration, and degree), immunological (clinical history of autoimmune diseases and presence of related organ-specific autoantibodies), and radiological features (neurohypophyseal bright spot, pituitary stalk thickening, and empty sella) in a large cohort of patients with apparently idiopathic CDI or CDI of known etiology. To this purpose, 150 patients with CDI were studied: 64 idiopathic, 6 familial, 12 associated to granulomatous diseases, and 68 secondary to cranial trauma, tumor, or surgery. AVPcAb were measured by an indirect immunofluorescence method. AVPcAb were found in 23.3% of CDI patients: 21 idiopathic (32.8%) and 14 nonidiopathic (16.3%; chi(2) = 13.1; P < 0.001). AVPcAb were independently associated with age less than 30 yr at disease onset (P = 0.001) in patients with idiopathic CDI and with history of autoimmune diseases (P = 0.006 and P = 0.02, respectively) and radiological evidence of pituitary stalk thickening (P = 0.02 and P = 0.003, respectively) in both idiopathic and nonidiopathic CDI. The likelihood of autoimmunity in one patient with apparently idiopathic CDI with age of disease onset less than 30 yr was 53%, it increased to 91% when history of autoimmune diseases was associated and to 99% when pituitary stalk thickening was further associated. In conclusion, autoimmunity is associated with one third of patients with apparently idiopathic CDI, which should therefore be classified as autoimmune CDI. Autoimmune CDI is highly likely in young patients with a clinical history of autoimmune diseases and radiological evidence of pituitary stalk thickening. Conversely, autoimmunity probably represents an epiphenomenon in patients with nonidiopathic CDI.

Inducible nitric oxide synthase is responsible for nitric oxide release from murine pituicytes.

This study investigated whether pituicytes were able to produce and release nitric oxide (NO), and which type of nitric oxide synthase (NOS) would be responsible for this phenomenon. Lipopolysaccharide (LPS) 1 micro g/ml was used as inflammatory mediator. Because pituicytes are known to secrete interleukin (IL)-6 upon stimulation with LPS, this parameter was also investigated. Cultured pituicytes, from 4-week-old male mice, were stimulated with LPS for 6 h or 24 h. At 24 h, there was a significant increase in accumulated nitrite indicating NO formation. In contrast, IL-6 release was already significantly higher 6 h after stimulation and further increased at 24 h. The correlation between accumulated nitrite and secreted IL-6 was 0.84 after 24 h of incubation with LPS. The expression of inducible NOS (iNOS) mRNA in the pituicytes was significantly higher than the control level after 6 h and 24 h of exposure to LPS, with levels at 6 h being significantly higher than those at 24 h. There was no detected expression of endothelial NOS or neuronal NOS mRNA. Cultured pituicytes were also subjected to immunocytochemistry for iNOS protein at 6, 12, and 24 h after stimulation with LPS. Most cells were positive for iNOS, but there were no observable differences with the time points that we used. Collectively, these results show that pituicytes are able to produce NO, and that the inducible form of NOS is responsible for this production. Furthermore, there is a weak correlation between NO and IL-6 released from pituicytes after 24 h of stimulation with LPS.

Double immunocytochemistry of vasoactive intestinal peptide and cGnRH-I in male quail: photoperiodic effects.

Reproduction in Japanese quail is primarily regulated by photoperiod. Vasoactive intestinal peptide (VIP) has been suggested as a transducer of environmental information, especially photoperiodic cues, to the hypothalamo-pituitary-gonadal axis. To investigate the possible interaction of VIP and the reproductive (gonadotropin-releasing hormone, GnRH) system, double-immunocytochemical staining for VIP and cGnRH-I was conducted in sexually mature male quail held under a long-day photoperiod (16L:8D; LD) and in sexually quiescent males held under a short-day photoperiod (8L:16D; SD). VIP-immunoreactive (ir) cells were found primarily in three locations: lateral septal organ (LSO) in nucleus accumbens (Ac), ventral hypothalamus, and infundibular area. VIP-ir cells in LSO displayed characteristics typical of cerebrospinal fluid (CSF)-contacting cells, and co-existed with cGnRH-I-ir cells and beaded fibers. In contrast, VIP-ir cells in the infundibular area did not co-exist with cGnRH-I-ir structures. The number of visible VIP-ir cells in the infundibular area of SD males was significantly lower than that of LD males, while the number of visible VIP-ir cells in Ac/LSO was not altered by photoperiod. A cluster of cGnRH-I-ir cells in the caudalmost septal area was heavily innervated by VIP-ir fibers, which appeared to contact cGnRH-I-ir cells directly at this location. Both VIP- and cGnRH-I-ir fibers heavily innervated the external layer of the median eminence (ME). These data suggest that Ac/LSO, the caudalmost septal area, and ME are possible sites of interaction between the VIP and the GnRH systems.

A longitudinal study of vasopressin cell antibodies, posterior pituitary function, and magnetic resonance imaging evaluations in subclinical autoimmune central diabetes insipidus.

Cytoplasmic autoantibodies to vasopressin-cells (AVPcAb) have been detected not only in patients with overt central diabetes insipidus (CDI), but also in patients with endocrine autoimmune diseases without CDI. This suggests that complete CDI can be preceded by a preclinical stage. Among 878 patients with endocrine autoimmune diseases without CDI, 9 patients found to be AVPcAb positive and 139 AVPcAb-negative controls were enrolled in this open prospective study. They were evaluated for AVPcAb and posterior pituitary function at least yearly for about 4 yr (range, 37-48 months); during this span, magnetic resonance imaging (MRI) of posterior pituitary and stalk was performed only in the AVPcAb-positive patients. Five of the 9 AVPcAb-positive patients had normal posterior pituitary function at study entry. They were AVPcAb positive throughout the follow-up period. At later stages of the study, 3 of them developed partial CDI, and 1 developed complete CDI. The remaining 4 patients showed impaired response to the water deprivation test at study entry and were diagnosed as having partial CDI. Two of them agreed to receive desmopressin replacement for 1 yr. After this treatment, the patients became negative for AVPcAb and displayed normal posterior pituitary function until the end of the follow-up. Conversely, the 2 untreated patients with partial CDI remained AVPcAb positive. One of them developed overt CDI. None of the controls became AVPcAb positive or developed CDI. The normal hyperintense MRI signal of the posterior pituitary, present at study entry, persisted subsequently in all 9 AVPcAb-positive patients, including those developing overt CDI, only disappearing in the late phase of complete CDI. In asymptomatic subjects, the monitoring of AVPcAb, but not MRI, seems to be useful to predict a progression toward partial/overt CDI. Early desmopressin therapy in patients with partial CDI could interrupt or delay the autoimmune damage and the progression toward clinically overt CDI.

A case of lymphocytic infundibuloneurohypophysitis: histophathological studies.

A 57-year-old woman presented with 2-year history of polyuria and polydipsia. Hormonal studies revealed almost normal anterior pituitary function and central diabetes insipidus. Magnetic resonance imaging showed thickening of the pituitary stalk and enlargement of the neurohypophysis without high intensity of the posterior lobe on T1-weighted images, which were compatible with lymphocytic infundibuloneurohypophysitis. Transsphenoidal biopsy was done and histological examination disclosed moderate fibrosis and lymphocytic infiltration not only in the posterior pituitary, but also in the adjacent anterior pituitary part of the gland. The lymphocytes both in the anterior and posterior pituitary were mainly T cells that were positive for UCHL 1, CD 3, and CD 8. Immunofluorescence of frozen tissue detected immunecomplex deposition in small vessels and the interstitium. These findings suggested that allergic reactions may play an important role in the pathogenesis of lymphocytic infundibuloneurohypophysitis.

Development of microglia and macrophages in the postnatal rat pituitary.

Microglia and macrophages, immunolabelled with F4/80 (which binds a 160-kDa plasmalemmal glycoprotein) and OX-42 (which labels the complement type 3 receptor, CR3), were identified in the neuro- and adenohypophyses, respectively, of postnatal rats from day 1 to adulthood. In the neurohypophysis, the numerical density (cells/mm2) of microglia increased from postnatal day 1 to day 7 but was then unchanged from the adult density. In the adenohypophysis, the numerical density of macrophages increased from postnatal day 1 to day 21. The increasing size of the pituitary meant that the total number of such cells increased rapidly in the neurohypophysis up to day 14, but was then essentially unchanged; in the adenohypophysis macrophages increased in proportion to the increasing size of the gland up to day 21. Proliferation of the mononuclear cells was analysed by the immunodetection of bromodeoxyuridine incorporation into the nuclei of microglia and macrophages. F4/80-immunoreactive cells incorporating bromodeoxyuridine were found on all the postnatal days studied. The proportion of such cells in the neurohypophysis was high from postnatal day 1 to day 14 and in the adenohypophysis was maximal on day 14, decreasing in both parts of the pituitary by day 21. The estimated total number of proliferating cells was maximal in both parts of the pituitary on day 14. In both parts, OX-42-immunoreactive cells were less numerous than F4/80-immunoreactive cells up to postnatal day 14; CR3 expression may therefore be associated with maturation of these cells. Neurohypophysial microglia increased in size to postnatal day 7, consistent with the assumption of a 'compact' microglial morphology; adenohypophysial macrophages did not change in size over the postnatal period. Throughout the period studied, neurohypophysial microglia were significantly more densely distributed and larger in size than adenohypophysial macrophages. Neurohypophysial microglia phagocytose terminals of neurosecretory neurons from day 7, concurrent with the development of a distinct perivascular space. In the adenohypophysis, the perivascular space was present from birth and macrophages were not phagocytic. There are, therefore, considerable differences in the density, morphology and activity between the populations of myelomonocytic cells in the postnatal rat neuro- and adenohypophyses.

In vitro effects of CINC/gro, a member of the interleukin-8 family, on interleukin-6 secretion by rat posterior pituitary cells.

We investigated the effects of CINC/gro on IL-6 secretion by rat posterior pituitary cells. CINC/gro immunoreactivity was already detected in 1-h conditioned medium of normal posterior pituitary cells, and it increased significantly in a time-dependent manner during the first 24 h of culture. This immunoreactivity could be induced by TNF-alpha in a dose-dependent manner. On the other hand, CINC/gro stimulated IL-6 secretion by posterior pituitary monolayer cultures in a concentration dependent manner. Thus, CINC/gro significantly (P < 0.01) increased the secretion of IL-6 within 13 h of incubation, and this effect continued throughout 24 h of incubation. The stimulatory effect of 100 ng/ml CINC/gro on IL-6 secretion was completely blocked by 24-h incubation with 100 ng/ml IAP. These data demonstrate a new biological activity for CINC/gro in the posterior pituitary system.

Microglia in the rat neurohypophysis increase expression of class I major histocompatibility antigens following central nervous system injury.

An immunocytochemical study of the expression of major histocompatibility complex (MHC) class I and II antigens by glial cells of the rat neurohypophysis was performed. Numerous cells with the appearance of microglia were found to constitutively express class I MHC antigens, while only rare cells expressed class II (Ia) antigens. Stereological analysis revealed that expression of class I MHC antigens increased significantly within 10 days after a unilateral hypothalamic lesion known to cause axonal degeneration and compensatory collateral axonal sprouting within the neurohypophysis. In addition, however, a brain lesion which did not affect the axonal population of the neurohypophysis also produced a significant increase in microglial expression of class I MHC antigens in this structure. Neither lesion affected the expression of class II MHC antigens in the neurohypophysis. Simultaneous immunofluorescent labeling for MHC I antigens and glial fibrillary acidic protein (GFAP, a pituicyte marker) or for MHC I and the C3bi complement receptor (a microglial marker) confirmed that the MHC class I-reactive cells were microglia. MHC I-positive cells also bound Griffonia simplicifolia B4 isolectin (GSA I-B4), consistent with their identification as microglia. The majority of MHC class I-reactive microglia were located in close apposition to blood vessels. These results indicate that an unusually large proportion of microglia within the neurohypophysis constitutively express MHC I antigens. In addition, neurohypophysial microglia are capable of responding to penetrating brain injury by upregulation of MHC I antigens in the absence of local tissue degeneration, possibly because of the absence of a blood-brain barrier.

Insulin-like immunoreactivity (IRI) in the rat hypothalamo-neurohypophysial system: effect of dehydration and haemorrhage.

Hypothalamic IRI was not affected in haemorrhaged rats, but diminished considerably in the dehydrated ones. In the neurohypohysis, IRI was distinctly higher both in dehydrated and haemorrhaged rats, i.e., under disorders which stimulated vasopressin and/or oxytocin release. It is suggested that insulin-like substance(s) may be someway involved in regulation of vasopressin or oxytocin secretion.

An immunohistochemical study of human pituicytes demonstrating frequent expression of MHC class II antigens and macrophage markers.

Using a panel of antibodies (Abs) and a lectin, normal human adult pituicytes were studied in neurohypophyses obtained from 29 patients at autopsy. The pituicytes reacted frequently with Abs against major histocompatibility complex (MHC) class II antigens (Ags), macrophage markers (KP.1, PG.M1, LN-5), an anti-vimentin Ab and a biotinylated lectin Ricinus communis agglutinin (RCA-1). The number of pituicytes immunostained for these reagents varied, with the notable exception of vimentin. MHC class II Abs (LN-3, CR3.43)-positive pituicytes were numerous in approximately half. Microscopically, MHC class II Ag was found in pituicytes of various shapes, and were identified in macrophage-typed pituicytes by electron microscopic immunohistochemistry. Glial fibrillary acidic protein was found in only a small number of pituicytes and was absent in cells labeled with MHC class II Abs or macrophage markers. The results indicate that the immunophenotype of human pituicytes is distinct from other glial cells of the central nervous system, with a considerable number of cells expressing MHC class II Ags and macrophage markers.

[The central role of the thymus in the education of T cells to neuroendocrine principles]. / Le rôle central du thymus dans l'éducation des cellules T aux principes neuroendocrines.

The thymus, the lymphoid organ responsible for the induction of central T cell self-tolerance, is the site of expression of peptides belonging to the neurohypophysial peptide family. The classical model of neurosecretion established by the Scharrers for the hypothalamo-neurohypophysial axis however cannot be applied to characterize the secretory pathways of neurohypophysial-related peptides in the thymic epithelial component. The novel model of cell-to-cell cryptocrine signalling has recently been proposed by J.W. Funder to describe the molecular relationships between fixed epithelial cells and migratory differentiating cells. In the thymus, the cryptocrine signalling is further closely associated to the presentation of the "self" molecular structure by major histocompatibility complex-derived proteins to developing T cells. On the basis of our observations, the model of the thymic repertoire of neuroendocrine "self" antigens transposes to the molecular peptide level the dual physiological role of the thymus in T cell negative and positive selection. Moreover, this model should also contribute to a better understanding of the molecular mechanisms underlying the central T cell tolerance of "self" neuroendocrine functions.

Ultrastructural distribution of glutamate immunoreactivity within neurosecretory endings and pituicytes of the rat neurohypophysis.

An ultrastructural analysis of post-embedding glutamate immunocytochemistry within the neural lobe of the pituitary was used to explore the possible role of glutamate within the magnocellular neuroendocrine cells. Relative densities of a colloidal gold marker associated with various cellular and subcellular compartments of the neural lobe were quantified by computer analysis of electron micrographs. Robust glutamate immunoreactivity was observed in both pituicytes (cytoplasm, mitochondria and nucleus) and neurosecretory endings. Within the neurosecretory endings, glutamate staining was specifically localized to the microvesicles with no overlap into the neurosecretory granule population. Stimulation of the vasopressin/oxytocin neurosecretory system by water deprivation increased glutamate content in pituicytes and mitochondria within neurosecretory endings but had little influence on microvesicle glutamate content. The results are consistent with the existence of multiple functional pools of immunoreactive glutamate in both pituicytes and neurosecretory endings. Microvesicles within the neurosecretory endings exhibit many properties of secretory vesicles, appear to be functionally independent of the neurosecretory granules, and have sufficient glutamate immunoreactivity to suggest that this amino acid may be compartmentalized for release in the neural lobe.

Depletion of CD8+ T cells suppresses growth of Trypanosoma brucei brucei and interferon-gamma) production in infected rats.

Sprague-Dawley rats infected with Trypanosoma brucei brucei showed a strong and rapid induction of splenocyte IFN-gamma (within 12 h post-infection) as measured by a single cell assay for IFN-gamma secretion. Depletion of CD8+ cells in infected rats abrogated the IFN-gamma production, suppressed parasite growth and increased survival of the animals. Induction of MHC class I antigens in the paraventricular and supra-optic hypothalamic nuclei caused by the trypanosome infection was also inhibited by the CD8+ cell depletion. It is suggested that the CD8+ cells are involved directly or indirectly in growth regulation of the parasite and that IFN-gamma induced by the parasite may be one of the factors that trigger MHC expression and immunosuppression.

Development of a novel screening device permitting immunocytochemical screening of numerous culture supernatants during hybridoma production.

A novel screening device is described which permits the simultaneous immunocytochemical processing of several hundreds or even thousands of hybridoma culture supernatants. The core of the screening apparatus is a foam-coated polymer plate that carries a 96-well pattern representing a modification of the actual 96-well template. This modification permits the use of conventional 26 X 76 mm microscopy glass slides. Each of these slides carries 24 carefully arranged histological sections. One 96-well plate is thus screened by mounting four of these slides in the apparatus during the primary antibody (i.e., culture supernatant) incubation stage. At all other stages of the immunocytochemical protocol, the slides are processed in the classical way. The screening apparatus has been used during the production of monoclonal antibodies against chicken pituitary glycoprotein hormones and against bovine neurohypophyseal peptides. In both instances, it proved to be the major contributory factor in the successful production of antibodies.