Proopiomelanocortin mrna expression by small cell bladder carcinoma associated with cushing’s syndrome: diagnostic contribution of in situ hybridization

A case of ectopic ACTH secretion by primary small-cell carcinoma of the urinary bladder is described. The patient, a 38-year-old woman, presented with Cushing's syndrome. Although immunocytochemistry of the tumor for ACTH and corticotropin-releasing factor yielded negative results, in situ hybridization demonstrated a conclusive signal for proopiomelanocortin messenger RNA. This case report shows the value of in situ hybridization in diagnostic pathology.     

Cytomegalovirus adrenalitis in acquired immunodeficiency syndrome

Seventy-three adrenal glands of 44 patients with acquired immunodeficiency syndrome (AIDS) were examined and graded histologically to reveal cytomegaloviral (CMV) adrenalitis. The number of CMV inclusion bodies (IB) were evaluated and compared with 3 methods in 58 adrenal glands of 40 patients: histological sections, immunocytochemistry for early antigens of CMV, and in situ hybridization with biotinylated probes for CMV DNA All 73 adrenal glands contained foci of lymphocytic infiltrate. Forty (55%) showed CMV adrenalitis and necrosis, which were more extensive in the medulla than in the cortex. The number of CMV IB increased with the severity of necrosis and fibrosis (grades I, 1.0; II, 3.6; III, 27.8 IB/ thousand cells counted in 20 fields). More than 85 percent of both glands were necrotic in0 only I patient (2.3%). For the 3 methods, the means of the number of CMV IB were as follows: in situ hybridization with biotinylated probe, 17.7; immunocytochemistry, 12.9; and H&E, 8.1. However, using multivariant analysis, there was no statistically significant difference. Thirty-three (45%) adrenal glands contained no CMV IB by any of the 3 methods. We conclude that CMV adrenalitis is a common finding in patients with AIDS. Destruction of adrenal tissue is usually not widespread enough to result in adrenocortical insufficiency.     

The neurofibromatosis gene in human pituitary adenomas

The neurofibromatosis 1 (NF1) gene encodes a protein neurofibromin, which contains a glutamyl transpeptidase (GTP)-activating protein (GAP)-related domain: NF1 GRD. This domain is able to down-regulate P^21ras by stimulating its intrinsic GTPase. Because P^2lras has an important role in regulating growth and differentiation, somatic mutations in the NF1 gene may result in mutant neurofibromins that might interfere with the Ras signaling pathway and contribute to the development of tumors. In this study, we used polymerase chain reaction (PCR)-coupled single-stranded conformational polymorphism (SSCP) and DNA sequencing to examine possible mutations in the NF1 GRD in human pituitary tumors. We screened 36 nonfunctioning and 20 growth hormone—secreting adenomas. No mutation was detected in these tumors. Our results indicate that inactivation of neurofibromin may not have a primary role in the formation of pituitary adenomas.     

Requirements for activation of CD8+Murine T cells

Cytolytic effector function fails to develop if proliferation of allospecific cytolytic T lymphocyte precursors is inhibited, but the requirements for generation of cytolytic activity have not been fully defined. In contrast, the cytolytic effector function of cytolytic T lymphocyte clones does not change during the cell cycle, and the level of cytolytic activity is independent of cellular proliferation. The requirement for proliferation by primary responding populations may reflect the need for clonal expansion of a few inherently cytolytic effector cells in order to reach a threshold number which can readily be detected in conventional cytolytic assays. Alternatively, proliferation may be required for cytolytic T lymphocyte precursors to differentiate into mature, functional cytolytic cells. Using CD8+T cells which express an antigen-specific transgenic α/β T cell receptor, we have studied the requirements for acquisition of cytolytic capacity. Stimulation of the T cell receptor alone appears to be sufficient to render naive, CD8+ transgenic T cells sensitive to the growth effects of interleukin-2 (IL-2), and in some circumstances to interleukin-4 (IL-4), but not to induce either lymphokine production or cytolytic activity. Costimulatory molecules expressed by allogenic stimulating cells appear to be required for lymphokine production, and CD8+ transgenic T cells initially appear to secrete only IL-2 and interferon-γ. Stimulation of the T cell receptor of naive, CD8+ transgenic T cells appears to induce cytolytic activity only if cell proliferation occurs, either in response to IL-2 produced by the stimulated cells themselves when costimulatory molecules are present, or to IL-2 or IL-4 from exogenous sources if costimulatory molecules are absent.     

Immunotherapeutic strategies targeting rheumatoid synovial T-cell receptors by DNA inoculation

Immunotherapy against autoreactive T-cell receptors (TCRs) has been reported to have promise in several animal models of autoimmune diseases. Facilitated DNA inoculation has many potential advantages as a modality for development of specific immune responses. Specifically, this technology is able to deliver exogenous antigens for processing via both the endogenous pathway, with subsequent presentation by class-I major histocompatibility (MHC) antigens, and the exogenous pathway, with subsequent presentation by class-II MHC antigens. This allows for induction of both arms of the cellular immune system. These cellular immune responses may be particularly important in targeting and controlling pathogenic cell populations. The application of this technology to the treatment of human autoimmune diseases depends on the availability of readily manipulated systems for the evaluation of specific interventions. Here we report the full length cloning and expression of TCRs from rheumatoid arthritis synovial tissue. These were developed by recombinant polymerase chain reaction, cloning and retroviral transduction into a TCR-α/β-negative murine T-cell hybridoma. Reconstitution of CD3 expression was confirmed by flow cytometry. Similar constructs have been developed for TCR-based immunotherpay by facilitated inoculation of DNA intramuscularly. Preliminary analysis of immune responses in mice indicates that these constructs elicit anti-TCR responses. These studies indicate the ability to reconstitute expression of potentially autoreactive human TCRs in a model system wherein specific immune responses elicited against these TCRs by various immunogenes can be evaluated.     

Detection of T cells responsive to a vascular growth factor in rheumatoid arthritis

The primary lesion in rheumatoid arthritis (RA) is a destructive synovitis characterized by proliferation of endothelial cells, fibroblasts, and vascular smooth muscle cells, and with perivascular lymphocyte aggregates. A nonhematopoietic growth factor, acidic fibroblast growth factor (aFGF), may induce many of the biological features found in rheumatoid synovium, including T cell activation. To determine if aFGF-responsive T cells are increased in RA, we developed an assay to measure the frequency of peripheral blood T cells that are costimulated by aFGF. The data indicate that the frequency of aFGF-responsive T cells is increased in RA and may change with disease activity and treatment.     

Granulocyte-macrophage colony-stimulating factor mimicry and receptor interactions

Development of small molecular mimics of larger polypeptide ligands is an important approach to pharmacophore design. One strategy for the development of such mimics is analysis of alternative ligands that bind to the same site as the native ligand. These allow examination of the structural and chemical constraints for binding within the setting of diverse backbone geometries. The use of antireceptor antibodies as alternative ligands has allowed the development of biologically active peptides in several ligand-receptor systems. This technology has been applied to the study of interactions between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFR). GM-CSF is one of a family of signal-transducing cytokines and growth factors characterized by a fourhelix bundle core structure. The GM-CSFR is comprised of an α-chain (GM-CSFRα) specific for GM-CSF, and a β-chain (β_c) shared with the interleukin-3 and interleukin-5 receptors. At least two sites on GM-CSF have been implicated in the GM-CSF-GM-CSFRα/β_c ternary complex. In studies summarized here, synthetic peptide analogs of GM-CSF sequences were designed and used to map neutralizing epitopes. One neutralizing epitope corresponded to the A helix of GM-CSF, and a synthetic analog displayed biological activity as a GM-CSF antagonist in vitro, suggesting interaction with the GM-CSFRα/β_c complex. A second peptide comprising the B and C helices was recognized by monoclonal neutralizing antibodies and similarly displayed antagonist activity. Recombinant antibody (rAb) technology was also employed. An expression library of rAbs from mice immunized with neutralizing anti-GM-CSF antibodies was developed and screened with a neutralizing anti-GM-CSF monoclonal antibody. One clone which displayed receptor binding activity exhibited structural similarity with epitopes on GM-CSF previously implicated as interaction sites with the neutralizing monoclonal antibody. A synthetic peptide analog of the rAb inhibited GM-CSF bioactivity. Critical contact residues were predicted on the basis of structural similarity of the rAb peptide and GM-CSF. These studies in dicate the feasibility of using rAbs in bioactive peptide design, providing lead compounds and information regarding contact residues for drug design.     

Amyloid deposits of the pituitary in old age: Correlation with histopathological alterations

The prevalence and quantification of amyloid formation and the frequency of histomorphological alterations affecting the pituitary gland were studied in a consecutive autopsy series performed on 109 patients older than 84 years of age; 87 (80%) pituitaries had amyloid in the anterior lobe. A polyclonal antibody directed against amyloid of A-light chain origin immunostained pituitary amyloid in every specimen, whereas all other antisera directed against the precursor proteins of the remaining major amyloid syndromes and pituitary hormones did not. Because no case studied suffered from a generalized A-light chain amyloidosis, immunostaining might be due to crossreaction with a hitherto unidentified precursor protein. Histomorphological alterations were observed in many pituitaries, and they were differentiated into regressive changes, cysts of the intermediate zone, so called basophilic invasion of the posterior lobe, hyperplasia, Erdheim’s squamous epithelia, and adenomas. Statistical analysis failed to reveal any correlations between the presence of histopathological alterations and the frequency or the amount of interstitial amyloid. Nonetheless, we were not able to explain amyloid formation in old age, especially none that was due to histomorphological alterations of the pituitary gland. Thus, other diseases that primarily do not affect the pituitary may actually influence pituitary amyloid formation.     

Modulation of T cell proliferative responses by accessory cell interactions

Antigen-specific activation of the T cell is accomplished by engagement of the T cell receptor (TCR) by an antigen (Ag)/MHC complex presented on the surface of an antigen-presenting cell (APC). However, it has been demonstrated that engagement of the TCR by Ag/MHC complexes alone is normally insufficient to lead to a proliferative response and the development of effector function. Thus it has been proposed that the APC also provides additional signals which serve to modulate the T cell's response. These second or costimulatory signals are thought to be critical in the generation of a T cell-driven immune response. Several receptors have been proposed to be capable of serving as costimulatory receptors. Candidate molecules include CD28 and LFA-1 as well as other receptors. In this review the studies that we have performed to clarify the role of both LFA-1 and CD28 in providing costimulatory activity for T cell activation are discussed. In addition, we present evidence that under certain conditions, TCR signalling alone can be sufficient to lead to T cell proliferation.     

T cell receptor analysis in rheumatoid arthritis: What have we learnt?

Many clues point to a role for T lymphocytes in the pathogenesis of rheumatoid arthritis (RA), although the importance of these cells and their position within the rheumatoid pathogenic scheme remain unknown. Encouraged by data from animal models of T-lymphocyte-mediated autoimmunity, a major focus of research into the role of T lymphocytes in RA has been the usage of T cell receptor V genes in rheumatoid synovitis. Despite many methodologic problems, involving choice of patients and controls, choice of specimens, and technical factors, several conclusions can be drawn from the published research. In particular, synovial T lymphocyte populations, as a whole, frequently show biased V gene usage and restricted clonality within those T lymphocyte subsets that utilize over-represented V gene families. Continued research into these synovial T lymphocyte subsets should provide important insights into the pathogenesis of RA, particularly if solutions to the identified methodologic problems are implemented.     

DNA inoculation as a novel vaccination method against human retroviruses with rheumatic disease associations

There are a number of rheumatologic manifestations of human retroviral infections associated with human immunodeficiency virus type I (HIV-I) and the human T-cell leukemia virus type I (HTLV-I) including arthritis, Sjøgren's syndrome-like symptoms as well as other varied autoimmune phenomena. Infection with HTLV-1 may be directly involved in the etiology and/or pathogenesis of an arthritic condition similar to rheumatoid arthritis. We have been characterizing a new vaccination strategy against human retroviral infections, designated DNA inoculation. This procedure involves the intramuscular injection of DNA plasmids which express specific human retroviral antigens. This technique results in the development of humoral and cellular immune responses against these proteins. Specifically, this method has been successfully used to develop immune responses against HIV-I and HTLV-I. The availability of rat and rabbit infection models for HTLV-I, coupled with the successful development of immune responses in these animals after DNA inoculation with an HTLV-I envelope expressing plasmid, will allow the efficacy of this vaccination technique to be evaluated with protection against in vivo viral challenge as an endpoint.     

Cushing’s syndrome caused by an ectopic pituitary adenoma of the sphenoid sinus: Adrenal crisis after partial resections of the adenoma

A 60-year-old woman with an 8-year history of Cushing’s syndrome was evaluated. Biochemical data were consistent with those of Cushing’s disease. Plasma ACTH levels responded paradoxically to GnRH. MRI demonstrated a large tumor occupying the sphenoid sinus, which was enhanced by gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). The pituitary gland was normal in shape and was located in the sella turcica without dislocation. The pituitary gland and the sphenoid tumor could be distinguished by the obvious difference in their MRI intensities. Three consecutive partial resections of the sphenoid tumor were performed, but plasma ACTH and cortisol levels remained high just after the third operation. Histological studies revealed a chromophobe adenoma immunohistochemically positive for ACTH. However, adrenal crisis occurred 3 months after the third operation during reserpine administration ( 1.5 mg/day for approximately 2 mo) for the treatment of Cushing’s syndrome due to the residual tumor in the sphenoid sinus. Subsequent MRI showed no change in the tumor shadow, and the paradoxical response of plasma ACTH levels to GnRH remained unchanged. The fourth operation reconfirmed the existence of the ACTH-producing adenoma in the sphenoid sinus. There was no anatomical interaction between the sphenoid tumor and the pituitary gland, and, histologically, no tumor cells were present in the pituitary gland. These findings suggest that the tumor is an ACTH-producing ectopic pituitary adenoma arising from the sphenoid sinus. The patient has been in remission for 4 years on glucocorticoid replacement therapy. The factors responsible for the adrenal crisis were not well understood, although reserpine administration might have had some role.     

Molecular and structural analysis of nuclear localizing anti-DNA lupus antibodies

To determine the structure of three nuclear localizing lupus anti-DNA immunoglobulins (Igs) and to search for clues to mechanisms of cellular and/or nuclear access, their H- and L-chain variable region sequences were determined and subjected to three-dimensional modeling. Although the results indicate heterogeneity in their primary structures, the H chains are encoded by 3 members of the J558 V_H gene family with a common tertiary conformation that is not shared by a J558-encoded nonnuclear localizing anti-DNA control Ig. Furthermore, at least two of the Igs share a conformational motif in the H-chain CDR3, and all three Igs contain multiple positively charged amino acids in their CDRs, resembling nuclear localization signals that direct protein nuclear import. Notably, each V_H and V_K gene is also found recurrently among previously described autoantibodies. Molecular analysis further indicates that both germline-encoded and significantly mutated V genes can generate nuclear localizing anti-DNA Ig.     

Suppressin: An endogenous negative regulator of immune cell activation

We have recently identified a new suppressor molecule we named suppressin (SPN) that has all the characteristics of a global negative regulator of the immune system. SPN is a unique 63-kD monomeric polypeptide with a pI of 8.1 that is produced and secreted under basal conditions by murine splenocytes, human peripheral mononuclear cells, and hormone-secreting pituitary cells. The biological actions of SPN in vitro include the inhibition of mitogen-induced proliferation and immunoglobulin synthesis of lymphocytes and the suppression of interleukin-2-dependent CTLL-2 cell proliferation. In addition, SPN enhances natural killer cell activity by eliciting interferon-α and-β synthesis and secretion. SPN effects are reversible, nontoxic, and require the continuous presence of exogenous SPN. T lymphocytes stimulated with concanavalin A or phytohemagglutinin are more sensitive to SPN (90% inhibition) than are lipopolysaccharide-stimulated B cells (60% inhibition). SPN arrests lymphocytes in the G₀/G₁ phase of the cell cycle after reduction of their RNA, protein and DNA synthesis, suggesting that SPN inhibits the processes required for G₀ transition to G₁. SPN is found intracellularly in all unstimulated lymphocyte subsets, monocytes, and in phytohemagglutinin-activated T lymphocytes immunopositive for the low affinity interleukin-2 receptor. These results suggest that SPN may be a major negative regulator of cell proliferation in the immune system. All SPN-producing cell types are also sensitive to SPN. Collectively, the results of these experiments provide the foundations for a model in which SPN regulates lymphocyte proliferation in an autocrine and/or paracrine manner. Additional evidence supporting this hypothesis was provided by experiments showing that by blocking endogenous SPN activity in vitro with neutralizing anti-SPN antibodies unstimulated lymphocyte proliferation is induced. Lastly, SPN also inhibits the proliferation of leukemia and lymphoma cells in vitro, suggesting a potential role for SPN in regulating tumor cell proliferation.     

DNA measurement,proliferation markers,and other factors in pituitary adenomas

To assess the proliferative activity of pituitary adenomas, 36 surgically removed adenomas were studied by light microscopical parameters; mitotic count; expression of PCNA, Ki-67, cathepsin D, and EGF; and image cytometry. Three adenomas (9%) showed high, 11 (34%) medium, 17 (53%) moderate, and 1 (3%) low structural differentiation. In 10 adenomas (31%), no mitosis was observed. The average was 2.4 mitoses/100 HPF; the highest count was 7.1 mitoses/100 HPF. Eleven adenomas (33.3%) were PCNA-negative; in 20 adenomas (60.6%), between 0.05 and 3.9, and in 2 adenomas (6.0%), between 10.5 and 16.4 PCNA-positive nuclei were observed. Only a recurrent null-cell adenoma (9%) was Ki-67-negative. Three adenomas (9.1%) were EGF-negative, 28 (84.8%) showed up to 10% positive cells, and 2 (6.1 %) showed between 10 and 30% positive cells; 19 adenomas (68%) were cathepsin D-negative, including all endocrine-inactive adenomas. Half the adenomas had an euploid DMA stem line. Endocrine-inactive adenomas displayed a higher rate of euploid DNA stem lines than endocrine-active adenomas. The S-phase fraction varied between 2.97 and 28%, with a mean value of 14.4%. Half the adenomas showed an S-phase fraction of 11.65% or lower.     

Ki-67 is an indicator of progression of neuroendocrine tumors

No current histological or cytological indices can distinguish reliably malignant from benign tumors in neuroendocrine tumors, including pheochromocytomas, pancreatic endocrine tumors, and carcinoid tumors. We investigated immunohistochemically the expression of Ki-67 in 52 neuroendocrine tumors, including 17 pheochromocytomas, 9 pancreatic endocrine tumors, 23 carcinoid tumors, 2 neuroendocrine carcinomas (NEC), and 1 neuroblastoma with liver metastasis. Of the 52 tumors, distant metastasis was observed in 4 pheochromocytomas, 2 pancreatic endocrine tumors, 4 carcinoids, 2 NEC, and 1 neuroblastoma. We classified these tumors into 3 groups; Groups A, B, and C, depending on the number of Ki-67-positive cells counted under a 200 x magnified field. Expression of Ki-67 was extremely high in group A (> 50 labeled nuclei/field), moderately high in group B (20–50 labeled nuclei), and very low in group C (< 10 labeled nuclei). There was a significant correlation between expression of Ki-67 and tumor progression. The tumors in group A progressed rapidly with the worst outcome; the tumors in group B progressed slowly but with a bad outcome; and the tumors in group C had no metastasis and a good prognosis. Ki-67 is an excellent indicator to assess progression of neuroendocrine tumors.     

Metallothionein expression in normal,hyperplastic, and neoplastic thyroid follicular and parafollicular C cells using monoclonal antimetallothionein antibody E9

Metallothioneins (MTs) are a set of ubiquitous low molecular proteins with a high affinity for metal ions, such as zinc, copper, and cadmium. MT overexpression can be induced by these metal ions as well as by other endogenous and exogenous factors. In this study, normal, hyperplastic, and neoplastic thyroid tissues of both follicular and C-cell origin were immuno-histochemically investigated with a monoclonal antibody against I- and II-isoforms of MTs. MT immunoreactivity was demonstrated in the follicular epithelium of 19 normal thyroid glands and in all 32 cases of Graves’ disease investigated; 26 of 30 follicular adenomas and 25 of 28 follicular carcinomas showed MT immunoreactivity, whereas only 7 of 20 papillary carcinomas were MT-positive (p < 0.0001 ). In 3 of the 7 positive samples, positivity was restricted to follicular areas of differentation. No MT could be immunolocalized in normal and hyperplastic C cells and medullary thyroid carcinomas (n = 20). In mixed medullary-follicular carcinomas (n = 4), MT staining patterns resembled those seen for thyroglobulin. In anaplastic carcinomas, MTs were mainly immunolocalized in nonspindle cell areas. MT expression in thyroid tumors may reflect the different biological behavior of follicular and papillary carcinomas. Antibodies to MTs may also serve as fairly specific immunohistochemical markers of follicular cell differentiation in thyroid neoplasia.     

Suppressive effect of intravenous immunoglobulins on the activity of interleukin-1

In order to study the effect of human immunoglobulin preparations for intravenous use (IVIg) on the production and activity of interleukin-1 (IL-1) derived from monocytes, we treated cultured monocytes with IVIg and examined the lymphocyteactivating factor (LAF) activity of IL-1 in the culture supernatants. The results showed that IVIg suppressed the activity from most healthy adults and some febrile children with acute respiratory disease or Kawasaki disease. Further studies revealed that intact Ig (whole molecular Ig) did not suppress the mRNA expression of IL-1α or IL-1β in mononuclear cells, that intact Ig and pepsin-digested Ig inhibited the LAF activity of recombinant IL-1 (rIL-1) and also that intact Ig contains immunoglobulin (probably anti-IL-1 antibody) which binds with rIL-1 by dot blotting using biotin-streptavidin. These results suggest that IVIg suppresses neither IL-1 synthesis nor the release of IL-1 from monocytes but does neutralize IL-1α and IL-1β activity by binding IL-1 proteins as an anti-IL-1 antibody.