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Summary: Background: Chondromalacia of the cranial medial femoral condyle (CMFC) is a potential cause of stifle lameness in adult horses. However, there is scant published evidence of either its occurrence or its clinical significance. Objectives: To document the occurrence of CMFC seen during diagnostic arthroscopy in adult horses with stifle lameness and to investigate its prognostic significance. Study design: Retrospective cohort study. Methods: The records were reviewed of all horses with unilateral or bilateral lameness localised to the stifle that underwent diagnostic arthroscopy of the cranial medial femorotibial joint at a UK equine hospital. The surgical findings were noted from each. Case outcomes were determined by unstructured telephone discussions with owners. A satisfactory outcome was defined as a horse that was in ridden work without ongoing anti‐inflammatory medication. Multivariable logistic regression was used to create a model with an outcome time point at 12‐month post‐operatively. Results: One hundred and four horses were included in the study. CMFC was found in 79. In 25 CMFC was the only finding, 54 horses had CMFC plus other pathology and 25 had other pathology, but no CMFC. At 12 months, horses with CMFC were 9.9 (95% CI 2.2–45.0, P<0.01) times more likely to have an unsatisfactory outcome than horses without CMFC. Main limitations: The study relied on retrospective analysis of clinical notes and archived arthroscopy videos. Assessment of outcome was determined by unstructured telephone interview and therefore there is potential for reporting errors to exist. Conclusions: CMFC is a common arthroscopic finding in horses with stifle lameness and is significantly associated with an increased likelihood of the horse not being in ridden work at long‐term follow‐up. [ABSTRACT FROM AUTHOR]

Keywords: horse; fracture; scintigraphy; standing surgery; racehorse EN horse fracture scintigraphy standing surgery racehorse 255 257 3 04/06/20 20200501 NES 200501 The case report by Cillán-García and colleagues published in this issue (Cillán-García I et al i . Conversely, the identification of a fracture on repeat radiography may have necessitated travelling the horse for surgical treatment at this later date, at which stage catastrophic fracture progression would be more likely. Treatment of the reported fracture by internal fixation is optimal to provide rigid stabilisation, interfragmentary compression and to minimise the risk of fracture propagation and secondary tarsometatarsal osteoarthritis. Fracture configuration, temperament of the animal, a surgeon experienced in fracture repair and a competent surgical team are all vital components of successful standing fracture repair. [Extracted from the article]

The interferons (IFN) are one of the body's natural defensive responses to such foreign components as microbes, tumors, and antigens. The IFN response begins with the production of the IFN proteins (alpha, beta, and gamma), which then induce the antiviral, antimicrobial, antitumor, and immunomodulatory actions of IFN. Recent advances have led to Food and Drug Administration approval of five clinical indications for IFN. Interferon alfa is approved for hairy-cell leukemia, condyloma acuminatum, Kaposi's sarcoma in the acquired immunodeficiency syndrome, and non-A, non-B (type C) viral hepatitis. Interferon gamma has properties distinctive from those of IFNs alpha and beta and is approved as an immunomodulatory treatment for chronic granulomatous disease. Promising clinical results with IFNs have also been reported for basal cell carcinoma, chronic myelogenous leukemia, cutaneous squamous cell carcinoma, early human immunodeficiency virus infection, hepatitis B, and laryngeal papillomatosis. Future clinical uses of IFNs may emphasize combination therapy with other cytokines, chemotherapy, radiation, surgery, hyperthermia, or hormones. [ABSTRACT FROM AUTHOR]

Murine mononuclear leukocytes express adrenocorticotropin (ACTH) receptors that were recognized by a monospecific antiserum to the ACTH receptor on Y-1 adrenal cells. The antiserum was utilized in an immunofluorescence (IF) assay to characterize the distribution of ACTH receptors on resting murine mononuclear leukocyte populations. Forty-seven percent of spleen cells, 32% of lymph node cells, and 1% of thymocytes constitutively expressed ACTH receptors. Separation of lymphocytes into purified B cell and T cell populations, followed by IF analysis revealed that 47% of B cells and 23% of T cells possessed ACTH receptors. Helper T cells (CD4+ T cells) constituted the majority of ACTH receptor-positive T lymphocytes. Furthermore, 47% of resident peritoneal macrophages, purified by adherence to plastic, expressed ACTH receptors. The T-lymphocyte mitogen, concanavalin A, interferon gamma, and ACTH enhanced ACTH receptor expression. The differential distribution of ACTH receptor-positive cells among specific leukocyte populations explains in part why differential cellular responses are observed and implies important regulatory functions for these receptors in the generation or regulation of immune responses.

To analyze the role of interleukin-10 (IL-10) in experimental autoimmune myasthenia gravis (EAMG) pathogenesis, we induced clinical EAMG in C57BL/6 and IL-10 gene-knockout (KO) mice. IL-10 KO mice had a lower incidence and severity of EAMG, with less muscle acetylcholine receptor (AChR) loss. AChR-immunized IL-10 KO mice showed a significantly higher AChR-specific proliferative response, altered cytokine response, lower number of class II-positive cells and B-cells, but a greater CD5(+)CD19(+) population than C57BL/6 mice. The lower clinical incidence in IL-10 KO could be explained not by a reduction of the quantity, but by a possible difference in the pathogenicity of anti-AChR antibodies.

Background: The transcription factor NF-kappaB plays a pivotal role in gene expression of inflammatory mediators such as cytokines or adhesion molecules. NF-kappaB-mediated transcriptional activation of these genes is inhibited by nitric oxide (NO) in a variety of cells, including monocytes. Morphine mediates NO release in a naloxone antagonizable manner in monocytes and neutrophils., Methods: The influence of morphine on NF-kappaB activation was investigated in a whole-blood flow cytometric assay. A specific antibody against the p65 subunit of NF-kappaB was used and detected by fluoresceine-isothiocyanate-labeled anti-immunoglobulin G. Nuclei were stained with propidium iodide. Leukocyte subpopulations were evaluated by gating on neutrophils and monocytes. The median fluorescence channel was determined. Different morphine concentrations (50 nm, 50 microm, 1 mm) and incubation intervals (10-150 min) were used., Results: Morphine inhibits lipopolysaccharide-induced NF-kappaB nuclear binding in human blood neutrophils and monocytes in a time-, concentration-, and naloxone-sensitive-dependent manner. Similar effects were achieved with the NO donor S-nitroso-N-acetyl-pencillamine and the antioxidant N-acetyl-cysteine. The NO synthase inhibitors Nomega-nitro-l-arginine-methyl-esther and Nomega-nitro-l-arginine completely abolished the morphine-induced attenuation of NF-kappaB nuclear binding, demonstrating that the inhibitory action is mediated by NO release., Conclusion: Morphine causes immunosuppression, at least in part, via the NO-stimulated depression of NF-kappaB nuclear binding.

Human herpesvirus 8 (HHV-8) has been causally linked to Kaposi's sarcoma (KS). There is significant homology between some HHV-8 genes and cellular genes including D-type cyclin (vCYC), G protein coupled receptor (vGCR), macrophage inflammatory proteins (vMIP-I, vMIP-II), bcl-2 (vBCL2), interferon regulatory factor-1 (vIRF1), interleukin-6 (vIL6), and complement-binding protein (vCBP). In this study, we analyzed expression of these viral homologs and HIV-1 Tat by reverse-transcriptase polymerase chain reaction (RT-PCR) coupled with Southern blot hybridization in AIDS-KS (AKS) tissue, classic KS tissue(CKS), and peripheral blood mononuclear cells, and phorbol ester (TPA)-treated and untreated HHV-8 positive lymphoma cells (BCBL1). While vCYC (AKS 6 of 6; CKS 3 of 3), vMIP-I (AKS 5 of 6, CKS 3 of 3), vBCL2 (AKS 6 of 6; CKS 3 of 3), and vIRF1 (AKS 5 of 6, CKS 3 of 3) transcripts were detected in both AKS and CKS, vGCR and HIV-1 Tat were expressed only in AKS samples (vGCR: AKS 3 of 6, CKS 0 of 3; Tat: AKS 4 of 6, CKS 0 of 3). vMIPII, vCBP, and vIL6 expression were not detected in any KS samples. Since vGCR expression is limited to AKS, it is possible that vGCR is activated by HIV-1 Tat. These results suggest that HIV-1 Tat may contribute to AKS pathogenesis through the tumorigenic and angiogenic effects of vGCR., (Copyright 2000 Academic Press.)

Certain functional interactions between the nervous, endocrine, and immune systems are mediated by cytokines. The pro-inflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF) were among the first to be recognized in this regard. A modulator of these cytokines, IL-10, has been shown to have a wide range of activities in the immune system; in this review, we describe its production and actions in the hypothalamic-pituitary-adrenal (HPA) axis. IL-10 is produced in pituitary, hypothalamic, and neural tissues in addition to lymphocytes. IL-10 enhances corticotropin releasing factor (CRF) and corticotropin (ACTH) production in hypothalamic and pituitary tissues, respectively. Further downstream in the HPA axis endogenous IL-10 has the potential to contribute to regulation of glucocorticosteroid production both tonically and following stressors. Our studies and those of others reviewed here indicate that IL-10 may be an important endogenous regulator in HPA axis activity and in CNS pathologies such as multiple sclerosis. Thus, in addition to its more widely recognized role in immunity, IL-10's neuroendocrine activities described here point to its role as an important regulator in communication between the immune and neuroendocrine systems.

Disruption of the linkage among the immune, nervous, and endocrine systems may contribute to the pathology and symptoms of acquired immunodeficiency syndrome (AIDS). We investigated the role of human immunodeficiency virus (HIV) in altering these linkages via induction of corticotropin (ACTH) by lymphocytes. Cultured T lymphocytes (H9 cell line) were infected with HIV-1, after which ACTH production was measured and characterized at various time intervals by immunofluorescence and Western blotting. We report a coordinate expression of ACTH and p24 HIV core protein in H9 cells. Also, the kinetics of HIV-induced ACTH production by H9 T lymphoma cells are demonstrated using three different strains of HIV as well as UV-inactivated HIV. ACTH production corresponded with the appearance of p24 antigen and was maximal 35 days after infection. UV-inactivated HIV and the viral envelope protein, gp120, were also able to induce ACTH production in these cells, indicating that viral replication was not required for the ACTH induction. The HIV-induced ACTH was synthesized de novo and had the size and biological activity of pituitary ACTH. Inhibition of ACTH in HIV-infected lymphocyte cultures by anti-ACTH antiserum enhanced viral p24 expression. The significance of lymphocyte ACTH in AIDS is not clear, but these results suggest that it may restrict HIV replication and possibly infection.

Following treatment with interleukin-10 (IL-10), basal corticotrophin releasing factor (CRF) levels from rat hypothalamic median eminence (ME) were found to be increased. Our data show: (1) the specificity of stimulation of CRF through the use of recombinant IL-10 and its blockage by monoclonal anti-IL-10 antibody; (2) the requirement of NO in this process through the use of N(omega)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor; (3) the blockage of IL-10 stimulated NO production by anti-IL-10; and, (4) the presence of IL-10 transcripts in hypothalamic poly A+ mRNA. These results provide the first evidence of IL-10 acting in the ME to influence CRF levels and further support our earlier findings of a potential for IL-10 in the hypothalamic-pituitary axis.

Recurrent respiratory papillomatosis (RRP), usually confined to the nasopharynx, trachea, and larynx, occasionally can progress to extensive bronchopulmonary disease. Most cases of bronchopulmonary and laryngeal papillomatosis are cytologically benign and do not undergo malignant transformation; however, squamous cell carcinoma (SCC) can arise in RRP in the absence of known risk factors such as radiation and smoking. In this study, the authors investigated molecular genetic alterations occurring in a case of metastasizing SCC that arose in long-standing bronchopulmonary papillomatosis. Genomic DNA from tracheal papillomata, tracheobronchial papillomata, SCC of the lung, and a lymph node metastasis was extracted. The physical state of the human papillomavirus type 11 (HPV-11) DNA was investigated by two-dimensional gel electrophoresis. Molecular genetic alterations of the host genome were studied by direct sequencing of polymerase chain reaction-amplified gene fragments and restriction fragment length polymorphism (RFLP) analysis. Episomal and integrated forms of HPV-11 sequences were detected in histologically benign tumors, but only the integrated form of the viral DNA could be found in malignant tissue samples. Molecular genetic studies revealed that an allelic loss of the interferon-beta gene (IFNbeta-1) and an endogenous type of mutation of the p53 antioncogene were found only in the malignant lesions. Mutations were not observed in the ras, neu, or multiple tumor suppressor (MTS1/p16) genes in any specimens. The authors' data indicated that the p53 genetic mutation was associated with integration of HPV-11 in histologically malignant lesions. This association may promote a progressive genetic instability that can lead to the development and clonal expansion of malignant lesions in RRP.

We demonstrate the presence of both delta and mu opioid receptors on the endothelium of human saphenous vein and internal thoracic artery. Displacement analysis revealed that a variety of opioid peptides were found to be ineffective in displacing specifically bound 3H dihydromorphine and only delta2 ligands were effective in regard to 3H Ala2-met5 enkephalinamide (DAMA), indicating the presence of mu3 and delta2 opioid receptor sites, respectively. Confirming the presence of both mu and delta sites we demonstrated positive immunostaining with anti-delta and anti-mu receptor antibodies. Exposure of these vessels to DAMA significantly enhances granulocyte adherence (P<0.01) even in vessels 5 min later exposed to 10(-6) M morphine. Unlike morphine, DAMA did not stimulate nitric oxide from either blood vessel and human granulocytes. Additionally, DAMA preadministered before morphine exposure to the endothelium or granulocytes, inhibited the morphine-stimulated release of NO in a dose-dependent manner. The data indicate that opioid peptides and opiate alkaloids regulate endothelial function in an antagonistic manner thereby influencing the microvascular environment.

Some of the effects that high-dose anabolic steroid abuse have and could have on the interactions between the immune and neuroendocrine systems are reviewed. Considering the past demonstrations on the actions of normal steroids on endocrine and immune responses, it is apparent that pharmacologically high doses of both normal and derivatized androgens (anabolic steroids) could have a significant effect. Indeed, some of the pathologies attributed to anabolic steroid abuse point to disturbances in the intimate connection between neuroendocrine and immune function and interaction. We attempt to review both the direct and indirect effects of this abuse, not only on this interaction but also on certain immune functions in particular.

Interleukin-10 (IL-10) is described as a cytokine that exerts immune downregulating actions. In this regard, our study indicates that IL-10 activity on human saphenous veins is coupled to nitric oxide (NO) release. We demonstrated this phenomenon by using in vitro real-time amperometric measurement of NO levels in explanted human saphenous veins after IL-10 exposure. IL-10-induced NO release can inhibit the adherence of monocytes (75.7 +/- 15 cells/600 microm2 of endothelial surface) and granulocytes (65 +/- 18 cells/600 microm2 of endothelial surface) from control values (250-300 cells/600 microm2 of endothelial surface; p < 0.005). This inhibition is directly sensitive to NO synthase inhibition. The specificity of the IL-10 effects is shown by its sensitivity to antibody. In vivo measurement of IL-10 levels during and after cardiopulmonary bypass surgery indicated that they are higher at 6 h after skin closure (1,400 pg/ml) compared with levels found during surgery (300 pg/ml). We surmise that the postsurgical increase of IL-10 levels may be an immunoregulatory attempt to downregulate the diffuse inflammation that has been shown to be associated with cardiopulmonary bypass surgery.

1. The effects of interleukin-2 (rhIL-2) and interleukin-4 (rhIL-4) were investigated on gamma-aminobutyric acid (GABA)-induced inward currents on isolated, identified neurons of Lymnaea stagnalis L. (Mollusca, Gastropoda) by using a concentration clamp technique. 2. It was shown that the interleukins modified the GABA-induced inward current in an opposite direction: rhIL-2 (2-100 U/ml) decreased the peak value of IGABA in a dose-dependent manner, whereas rhIL-4 (0.2-100 U/ml), on the contrary, potentiated it. Both types of modulation were partially or fully reversible. 3. The reversal potential of IGABA was not shifted by these cytokines. 4. The time-to-peak value and inactivation time constant of the gamma-aminobutyric acid (GABA)-induced current was decreased by rhIL-4. The modulatory effect of rhIL-4 was eliminated after conjugation of this cytokine with its antibody. 5. It appears that cytokines could play a role in regulating the neural excitability through GABA-erg mechanisms.

Human papillomaviruses (HPVs) are double-stranded, circular, epitheliotropic DNA viruses of which nearly 70 types have been identified. Specific HPV types exhibit a predilection to infect certain sites; however, occurrence is not unique or restricted to these sites. HPV typing may also be helpful in determining the oncogenic potential of HPV lesions. The most common HPV types, 6 and 11, are associated with benign mucosal lesions, whereas types 18, 16, 31, and 33 are thought to confer a high rate of malignant transformation. We describe a patient with both palmar verrucae and esophageal papillomatosis that proved to be HPV type 45 by polymerase chain reaction. HPV 45 has a high homology to HPV 18 and is a member of the relatively new "high-risk" mucosal HPV family in terms of cervical oncogenic potential. To our knowledge, HPV 45 has never been reported in cutaneous warts or esophageal lesions.

This is the first study documenting the induction of gamma-interferon (IFN-gamma) in human embryonic fibroblasts during human cytomegalovirus (HCMV) replication. Infection of cells with HCMV resulted in the consistent production of IFN-gamma RNA, as determined by RT-PCR and Northern blot analysis. Western blot analysis of cell lysates and immunoprecipitates from the cultural fluids of infected cells demonstrated the presence of IFN-gamma at the protein level. Induction of IFN-gamma required infectious HCMV, since high-dose ultraviolet inactivation of the virus stock eliminated IFN-gamma production. Further, IFN-gamma induction appears to be a late event in the virus replication cycle, since inhibition of HCMV DNA synthesis (e.g., phosphonoacetic acid) blocked the increase in IFN-gamma. Soluble factor(s) released from HCMV-infected cells apparently did not contribute to the induction of IFN-gamma, since virus stocks from which virus had been removed by sedimentation did not induce production of IFN-gamma. The appearance of IFN-gamma at late stages of HCMV infection and its elimination in the presence of an inhibitor (Actinomycin D) of RNA synthesis indicate a true transcriptional induction of this lymphokine at the RNA and protein levels. The significance of IFN-gamma production with regard to the replication and pathogenesis of HCMV in vitro and in vivo will require further investigation.

Sleep is altered during the course of viral infection, including that in which the human immunodeficiency virus (HIV) is the etiologic agent. Alterations in the sleep of HIV-infected individuals occur early in the course of infection, prior to the onset of AIDS. The mechanisms for such alterations in sleep are not known. The HIV envelope glycoprotein 120 (gp120) induces the synthesis and secretion of cytokines that enhance [e.g., interleukin (IL)-1 and tumor necrosis factor] and suppress (e.g., IL-10 and IL-1 receptor antagonist) sleep. We used a well-defined rat model to test the hypothesis that the HIV gp120 alters sleep. Recombinant HIV-1IIIB gp120 was injected intracerebroventricularly (20- 500 ng) into rats prior to dark onset. Sleep-wake behavior was not altered after the 20-ng dose, whereas both non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) were initially enhanced and subsequently suppressed after the 100-ng dose. NREMS was enhanced for 8 h after the 500-ng dose; REMS was not affected by this dose. Brain temperature was not altered by any of the gp120 doses used in this study. In addition, mRNA expression for IL-1 beta and IL-10 was induced in the hypothalamus by gp120; this brain region is crucial for the regulation of sleep. These new data support the hypothesis that altered cytokine concentrations within the central nervous system play a pivotal role in the complex alterations in sleep observed during HIV infection.

Cardiopulmonary bypass (CPB) results in a diffuse inflammatory response characterized in part by hyperstimulation of leukocytes. We have previously shown that this hyperstimulation appears to be due, in part, to an increase in the release of biological response modifiers (BRMs) such as cytokines. In the present study, we evaluated the ability of a naturally occurring immunocyte inhibitory substance, alpha-melanocyte-stimulating hormone (alpha-MSH), to prevent the hyperstimulation caused by CPB. Monocytes and granulocytes were pretreated with alpha-MSH (10(-6) M) before exposing the cells to plasma obtained from patients who had undergone CPB, as CPB plasma would stimulate native monocytes and granulocytes in a manner similar to that observed in CPB patients. Pretreatment of these cells with alpha-MSH significantly diminished the hyperstimulation induced by CPB plasma in a concentration-dependent manner. In contrast, when the cells were first or simultaneously exposed to CPB plasma and then to alpha-MSH, alpha-MSH had no effect. Furthermore, use of the specific neutral endopeptidase inhibitor, phosphoramidon, significantly increased the efficacy of alpha-MSH in inhibiting CPB-induced immunocyte activation. The data demonstrate that pretreatment of monocyte/macrophages and granulocytes with alpha-MSH effectively inhibits the immune hyperstimulation induced by CPB-plasma exposure. In addition, the data strongly suggest that preexposure to other naturally occurring immune inhibitory substances may diminish the hyperstimulation associated with CPB. The study also further confirms that this hyperstimulation may, in part, be due to BRMs released from immunocytes.

The discovery of the ability of the nervous system to communicate through "public" circuits with other systems of the body is attributed to Ernst and Berta Scharrer, who described the neurosecretory process in 1928. Indeed, the immune system has been identified as another important neuroendocrine target tissue. Opioid peptides are involved in this communication (i.e., neuroimmune) and with that of autoimmunoregulation (communication between immunocytes). The significance of opioid neuropeptide involvement with the immune system is ascertained from the presence of novel delta, mu, and kappa receptors on inflammatory cells that result in modulation of cellular activity after activation, as well as the presence of specific enzymatic degradation and regulation processes. In contrast to the relatively uniform antinociceptive action of opiate and opioid signal molecules in neural tissues, the presence of naturally occurring morphine in plasma and a novel mu3, opiate-specific receptor on inflammatory cells adds to the growing knowledge that opioid and opiate signal molecules may have antagonistic actions in select tissues. In examining various disorders (e.g., human immunodeficiency virus, substance abuse, parasitism, and the diffuse inflammatory response associated with surgery) evidence has also been found for the involvement of opiate/opioid signaling in prominent mechanisms. In addition, the presence of similar mechanisms in man and organisms 500 million years divergent in evolution bespeaks the importance of this family of signal molecules. The present review provides an overview of recent advances in the field of opiate and opioid immunoregulatory processes and speculates as to their significance in diverse biological systems.

Opioid peptides activate immunocytes and opiate alkaloids inhibit this activation in the mussel, Mytilus edulis. Here we present evidence that cells of another invertebrate, Leucophaea maderae, can be influenced in a similar way by the Met-enkephalin analogue D-Ala2-Met5-enkephalin (DAMA) and morphine. Effects of different signal molecules on Leucophaea hemocytes were evaluated by computer-assisted image analysis of their conformational state. A small percentage of the untreated cells were found to display spontaneous conformational changes after 25 min of incubation without pharmacological agents which was noted as a decrease in both circularity factor and shape factor values. Activation caused the cells to become elliptical, a feature that appears to be characteristic of Leucophaea immunocytes. Administration of DAMA induced a similar activation of most of the cells. After 30 min these DAMA-activated cells started to display distinct locomotory activity not seen in the controls. alpha-Melanocyte-stimulating hormone (MSH, 10(-7)) added to the incubation medium after DAMA-activation caused the cells to return to their original "rounded" conformation. In addition, the presence of immunoreactive interleukin (IL-1), adrenocorticotropin (ACTH) and tumor necrosis factor (TNF) in the hemolymph was demonstrated. These data suggest an interaction between both vertebrate-type immunological signal molecules and neuropeptides in the regulation of immunological cells in Leucophaea.

Anabolic androgenic steroids (AS) have recently been placed on the Food and Drug Administration's (FDA's) list of controlled substances, because of the adverse effects seen in athletes taking accelerated dosages in attempts to enhance performance. Reported deleterious effects on abusers include sterility, gynecomastia in males, acne, balding, psychological changes, and increased risks of heart disease and liver neoplasia. Considering the roles of the immune and neuroendocrine systems and their interactions in many of these pathologies, it is important to determine the effects of these derivitized androgens on this connection. Little is known in this respect. We therefore determined the effects of anabolic steroids on certain immune responses and their effects on the extrapituitary production of corticotropin by lymphocytes. We present evidence that (1) both 17-beta and 17-alpha esterified AS, nandrolone decanoate and oxymethenelone, respectively, significantly inhibited production of antibody to sheep red blood cells in a murine abuse model; (2) the control androgens testosterone and dehydroepian-drosterone (DHEA) or sesame seed oil vehicle had no significant effects on antibody production; (3) nandrolone decanoate and oxymethenelone directly induced the production of the inflammatory cytokines IL-1 beta and TNF-alpha from human peripheral blood lymphocytes but had no effect on IL-2 or IL-10 production; (4) control androgens had no direct cytokine inducing effect; (5) nandrolone decanoate significantly inhibited IFN production in human WISH and murine L-929 cells; and (6) nandrolone decanoate significantly inhibited the production of corticotropin in human peripheral blood lymphocytes following viral infection. These data indicate that high doses of anabolic steroids can have significant effects on immune responses and extrapituitary production of corticotropin. Furthermore, the mouse model should provide an effective means by which to study other deleterious effects of anabolic steroid abuse in humans.

There is general agreement that IFN-gamma is produced only by cells of immune origin (T-cells, NK cells, and recently macrophages). However, indirect evidence has suggested that undetectable, low levels of IFN-gamma produced by cells of non-immune lineage, such as the murine line L-929, enhanced the antiviral activity of IFNs-alpha and/or beta following induction by agents such as the double stranded RNA poly ICLC. Since L-929 cells were one of the prototypic cell lines for studying murine IFN induction and action, we felt that it would be important to validate this observation by detection of the mRNA for IFN-gamma. If confirmed, it might indicate a role for IFN-gamma in non-immune cells. The present investigations revealed that mouse L-929 fibroblasts produce IFN-gamma message following exposure to conventional IFN-alpha/beta inducers such as poly ICLC or Newcastle disease virus. In addition, we found that IFN-gamma itself will induce its own message. We further show that this is not a phenomenon isolated to transformed cells since we found that normal mouse embryo fibroblasts also produced the message, however in a constitutive fashion.

Kaposi's sarcoma (KS) accounts for more than 15% of AIDS-related malignancies. The etiology of KS is unresolved but is postulated to be multifactorial, involving viruses and overexpression of cellular growth factors and/or oncogenes. Recently, herpesvirus-like sequences (KSHV) were identified with high prevalence in AIDS-KS (AKS), endemic KS, and in classic KS biopsies (CKS). To confirm the presence and the prevalence of the KSHV sequences, 18 CKS and 13 AKS samples were tested using polymerase chain reaction (PCR) analysis. To our knowledge this is the highest number of CKS samples that has ever been included in a single study, and it is also important that the biopsies were obtained from different institutions and geographical locations. KSHV sequences were detected in 100% of the AKS samples and 72% of the CKS biopsies using PCR analysis. The presence of the unique KSHV sequences was confirmed by direct sequencing of representative PCR products obtained from AKS and CKS samples. Reverse transcriptase (RT)-PCR experiments showed that the KSHV sequences were transcribed to mRNA in both AKS and CKS samples. Our results confirm that the putative new herpesvirus-like agent is associated with both AKS and CKS and may have an etiological role in the pathogenesis of this malignancy.

Objective: Our purpose was to determine whether interleukin-10 is expressed in human placental tissue, which might imply a role for it in fetal allograft protection., Study Design: Detection of interleukin-10 messenger ribonucleic acid in human placental tissue and in human placental JAR cells by reverse transcription-coupled polymerase chain reaction was studied., Results: Interleukin-10 messenger ribonucleic acid was detected in human placental tissue from term mothers and in human placental JAR cells. Sequence analysis of the expected interleukin-10 complementary deoxyribonucleic acid fragment revealed 100% homology to authentic interleukin-10 complementary deoxyribonucleic acid., Conclusion: Our results indicated that human placental tissue from term mothers expressed high levels of interleukin-10 messenger ribonucleic acid, suggesting that cells that produce interleukin-10 and that are associated with the placenta may play a role in preventing rejection of the fetal allograft by the mother.

Interleukin-10 (IL-10), originally designated a cytokine synthesis inhibitory factor, inhibits the synthesis of the pro-inflammatory cytokines IL-1 and tumor necrosis factor by stimulated human and mouse monocytes/macrophages; these cytokines are involved in the regulation of sleep. To determine if IL-10 reduces spontaneous sleep, we injected murine recombinant IL-10 intracerebroventricularly into rats prior to light onset. Non-rapid eye movements sleep was reduced. The behavioral responses to IL-10 were abolished by heat-inactivation of this cytokine. We believe these to be the first observations of central nervous system actions for this cytokine. These results further support the hypothesis that cytokines are involved in the regulation of sleep, and suggest an additional mechanism whereby sleep may be altered in response to an activated immune system.

Consensus degenerate primers specific for the L1 and E1 regions were used to amplify anogenital HPV-DNA fragments. The mixture of the viral fragments was then directly sequenced with HPV-6, -11, -16, -18 and -33 type-specific computer-designed oligonucleotides as sequencing primers. The linking of a consensus primer-generated PCR amplification with type-specific primer-mediated direct sequencing and computer data bank analysis provided precise, more objective viral detection in the simultaneous presence of different HPV types. The method was successfully adapted for viral typing in clinical lesions which simultaneously contained different anogenital HPV sequences.

Herpesvirus-like DNA sequences (KSHV) have been reported to be associated with various forms of Kaposi's sarcoma (KS). To determine if KSHV was associated with other proliferative skin lesions from non-AIDS immunocompromised patients, 33 skin lesions (basal cell carcinomas, squamous cell carcinomas, actinic keratoses, verruca vulgaris, atypical squamous proliferations, and seborrhoeic keratosis) from 4 organ-transplant patients receiving immunosuppressive therapy were tested for KSHV by PCR. KSHV sequences were detected in 82% of these skin lesions. Our results suggest that KSHV is associated with lesions other than KS in non-AIDS immunocompromised patients, and may also be involved in the pathogenesis of the various forms of proliferative skin lesions from organ-transplant patients.

1. Recent data have shown that interleukin-10 (IL-10) is expressed and acts in mouse pituitary tumor cells and freshly isolated mouse pituitaries. 2. In this study, we show that poly(A+) RNA derived from normal human pituitary and hypothalamus expresses IL-10 message. 3. The majority of transcripts was likely from the pituitary and hypothalamus, and not from lymphocytes in the pituitary and hypothalamic vasculature, since both IL-10 and interferon-gamma mRNA levels, compared to equivalent amounts of RNA from peripheral blood lymphocytes, were much lower. 4. These results indicate that IL-10 may function in human neuroendocrine process as it does in the murine system, thus serving as an important signal molecule for bidirectional communication between the neuroendocrine and the immune systems.

Objective: The trauma of cardiopulmonary by-pass (CPB) in cardiac surgery results in a whole body diffuse inflammatory response characterized in part by hyperstimulation of leukocytes. Partially this is due to an increase in the release of biological response modifiers such as cytokines, as noted by the immunocyte stimulatory actions of cell-free plasma obtained postoperatively from CPB patients. The present study was conducted to determine whether CPB plasma induced immunocyte hyperstimulation can be prevented with naturally occurring immune inhibitory substances, specifically, interleukin (IL)-10 and/or morphine., Experimental Design: Controlled in vitro study of the application of drugs to naive immunocytes to block the exitation caused by CPB-plasma., Setting: University-based tertiary care hospital., Patients: Plasma was obtained from ten patients undergoing CPB. Eligibility included admission for elective cardiac surgery, which no chronic illnesses or acute processes., Interventions: Monocytes and granulocytes were pretreated with IL-10 and/or morphine before exposure to plasma obtained from patients undergoing CPB, as CPB-plasma would stimulate naive monocytes and granulocytes in a manner similar to that previously reported in CPB-patients., Measures: Computer-assisted microscopic image analysis, measuring cellular conformational and velocity changes, was used to evaluate the effect of treatment on the immunocytes response to stimulation with CPB-plasma., Results: Pretreatment of cells with IL-10 and/or morphine significantly diminished the hyperstimulation induced by CPB-plasma in a concentration-dependent manner. In contrast, when the cells were initially or simultaneously exposed to CPB-plasma, IL-10 and/or morphine had no effect.

Tumor necrosis factor (TNF) is a monokine produced primarily by macrophages. TNF has a number of activities including direct lysis of certain transformed cells and induction of antiviral activity. One of the protoypical transformed cell lines used for studying TNF cytolysis is murine L-929 cells. Because of the lysis, TNF has not been shown to have antiviral activity in these cells. Since retinoic acid (RA) induces a normal phenotype in the L-929 cells, we sought to determine if their conversion to a normal phenotype would 1) render them insensitive to the cytolytic effect and 2) allow for the development of an antiviral state. We present evidence that both the cis- and trans- forms of RA and to a lesser extent, the RA precursor beta-carotene, can inhibit recombinant human TNF cytolytic activity in mouse L-929 cells. However, blockage of the cytolytic activity does not allow development of an antiviral state.

The effect of interleukin-4 (IL-4) was studied on voltage-gated Ca-current of the neuron RPeD1 of Lymnaea stagnalis L. (Mollusca, Basommatophora), using two-microelectrode voltage-clamp method. It was found that: 1. The neuron RPeD1 possessed a high voltage-activated Ca-current characterized by -40 mV activation threshold and reaching its maximum at +5+5(-)+10 mV. The activation time constant of the current was found to be a monnexponential function of the membrane potential. Currents were almost completely inactivated within 200-300 ms. 2. IL-4 (10-200 U/ml) uniformly and reversibly increased the peak value of Ca-current in a dose- and time-dependent manner (at concentrations 75, 150 and 200 U/ml the potentiation was 5.3 +/- 1.8% (n = 3), 5.5 +/- 1. (n = 3) and 14.6 +/- 7.4% (n = 4), respectively. 3. No changes in the membrane resistance and holding current were observed during IL-4 application. 4. The time constants of activation and inactivation were not affected by IL-4. The maximal conductance as the only kinetic parameter was increased under the influence of IL-4. 5. The affect in modulation of HVA Ca-current caused by IL-4 was additive between the doses of 10-15o U/ml but saturated at concentrations > 200 U/ml. 6. IL-4 antiserum was a potent inhibitor for enhancement of Ca-current by IL-4.

We have developed a novel method to study the subtype-specific expression of interferon-alpha (IFN-alpha) in the mouse system: we synthesized and used consensus oligonucleotide primers to allow simultaneous polymerase chain reaction (PCR) amplification of multiple murine IFN-alpha gene sequences. In addition, a set of subtype-specific oligomer probes were designed and used to distinguish between IFN-alpha genes that differ by only a few bases. The consensus primers, corresponding to two regions highly conserved among murine IFN-alpha subtypes, were used in reverse transcription and PCR amplification of total cellular RNA, isolated from IFN-gamma-treated murine L-929 cells, to yield a fragment of the anticipated approximately 520-bp size. Southern analysis of the amplified product using an internal consensus oligomer probe confirmed specific amplification of murine IFN-alpha gene(s). Subtype-specific oligonucleotide probes indicate that IFNs-alpha 1, -alpha 2, and -alpha 5 are present following IFN-gamma treatment, whereas IFN-alpha 4 remains virtually absent. Our results indicate that the expression of specific IFN-alpha subtypes may be subject to complex regulation, dependent upon inducing agents and cell types involved, and countless other factors. The procedure described here represents a novel method for studying the subtleties of the murine IFN-alpha mRNA expression.

1. Interleukin-10 (IL-10) has a wide range of activities in the immune system such as modulation of interferon-gamma (IFN-gamma) and antibody production. The neuropeptide hormone corticotropin (ACTH) has similar activities, suggesting that a bidirectional communication mechanism operates between the immune and the neuroendocrine system involving these two substances. 2. Murine pituitary tumor cells (AtT-20) were found to produce up to 3 ng/ml of IL-10. 3. Pituitary cell corticotropin production was enhanced by IL-10 treatment. 4. IL-10 induced the production of ACTH in mouse splenocytes. 5. Authenticity of pituitary-derived IL-10 was shown by the demonstration of identical nucleic acid sequences of reverse-transcribed, polymerase chain reaction amplified fragments of cDNA obtained from murine splenocytes, a murine pituitary tumor cell line, and freshly isolated murine pituitaries.

Consensus primers specific for the L1 and E1 regions were used to amplify HPV DNA fragments. These fragments were then directly sequenced using the consensus primers as the sequencing primers. The linking of PCR amplification, direct sequencing, and computer data bank analysis provides a new approach in the detection of different HPVs and has several advantages over existing methodology. These advantages include increased precision in the rapid characterization of known HPVs, detection of mutations, and identification of new HPV types.

The effects of a synthetic peptide fragment of human immunodeficiency virus gp120 (HIV gp120) on opioid (D-ala2-D-met5 enkephalinamide; DAMA) and interleukin-1 (IL-1) induced chemotactic responses in human granulocytes and monocytes and invertebrate (Mytilus edulis) immunocytes were studied. Both DAMA and IL-1 increased the velocity of cell migration from both species and the response is chemotactic (e.g. directed). Non-treated control cells move randomly or not at all. The addition of gp120 to DAMA or IL-1 treated human granulocytes or monocytes results in a slower movement which is chemokinetic (loss of directionality or random) in nature. A similar phenomenon occurs in the invertebrate immunocytes. If gp120 alone is added, it inhibits the movement of spontaneously active human granulocytes and Mytilus edulis immunocytes. In contrast, it stimulates chemokinesis of spontaneously active human monocytes. These responses occur immediately after addition of the peptide. Based on experiments with the selective calcium channel antagonist nimodipine, it appears that the gp120 causes its effects by irreversible binding to a calcium channel. Our results suggest a universal inhibitory mechanism is occurring since the invertebrate immunocytes must recognize HIV gp120 peptide to result in this effect, possibly through a CD4 or other type of surface determinant.

Neutral endopeptidase (NEP) 24.11 appears to be an important enzyme in both vertebrate and invertebrate autoimmunoregulation. Activation of human or invertebrate immunocytes that express NEP with substrates such as monokines and neuropeptides results in its increased expression, in other words, upregulation. However, since certain neuropeptides are also substrates for NEP, these activated immunocytes will respond to neuropeptides only at higher concentrations, thus downregulating the response. Specifically, in tumor necrosis factor (TNF)-treated immunocytes, we demonstrate the effects of increased NEP expression on altering the stimulatory activities of the neuropeptides met-enkephalin, melanocyte-stimulating hormone and substance P. We demonstrate the significance of NEP in modulating these responses through the use of specific enzyme inhibitors such as phosphoramidon, thiorphan and captopril. Furthermore, we present evidence suggesting that the individual variations seen in immunocytes from both different and the same donors to activating substances may reflect fluctuating levels of NEP expressed in response to endogenous stimuli. These results indicate that NEP is a highly significant factor in controlling the response(s) of certain immunocytes in man and higher invertebrates to the influence of biologically active substances such as monokines and neuropeptides.

The effects of the opioid neuropeptide [D-Ala2]deltorphin I, isolated from amphibian skin, on immunoregulatory activities were studied in representatives of vertebrates and invertebrates. The high potency of this compound parallels that of [Met]enkephalin, which was previously demonstrated in vertebrate plasma and invertebrate hemolymph. The addition of [D-Ala2]deltorphin I at 10(-11) M to human granulocytes or immunocytes of the mollusc Mytilus edulis resulted in cellular adherence and conformational changes indicative of cellular activation. This value is in line with the concentrations obtained with [Met]enkephalin, tested in the presence of the specific neutral endopeptidase 24.11 inhibitor phosphoramidon, and this opioid's synthetic analog [D-Ala2, Met5]enkephalin which, like [D-Ala2]deltorphin I, is resistant to proteolytic degradation. Both ligands appear to be acting on the same population of immunocytes. The same relationship was estimated to exist in the insect Leucophaea maderae, in which the high viscosity of the hemolymph makes the quantification of reactive cells more difficult than in Mytilus. In addition, [D-Ala2]deltorphin I is as potent as beta-endorphin in affecting the proliferation of lymphocytes in response to mitogen. Saturation experiments with unlabeled ligands and the radioligands [3H][D-Ala2]deltorphin I and [3H][D-Ala2,Met5]enkephalinamide revealed the presence of two high-affinity binding sites on human granulocytes, one sensitive to the nonequilibrium delta opioid antagonist [D-Ala2,Leu5,Cys6]enkephalinamide and the other relatively insensitive. The results obtained with [D-Ala2]deltorphin I support the view that the special role played by endogenous [Met]enkephalin in immunobiological activities of vertebrates and invertebrates is mediated by a special subtype of delta opioid receptor.

1. Mytilus pedal ganglion contains a small population of glial cells that are immunopositive for interleukin-1 alpha. Positively stained fibers can also be seen in the neuropil of these sections. 2. The marine worm Nereis diversicolor also exhibits positive neural immunostaining for interleukin-1 alpha. 3. Both organisms contain hemocytes that contain immunoactivity for interleukin-1 alpha. The study suggests interleukin-1 alpha to be an ancient cytokine given its presence in organisms that evolved significantly earlier than mammals.

1. Corticotropin-releasing factor (CRF) appears to be a potentially important signal molecule in both vertebrate and invertebrate neuroimmune and autoimmunoregulatory processes. It appears to mimic the effects of alpha-melanocyte stimulating hormone (MSH) but has a longer duration of action. 2. alpha-Helical CRF, a specific inhibitor of CRF, antagonizes CRF-induced cellular immunosuppression but is ineffective in altering MSH-induced immunosuppression. 3. Both human and Mytilus immunocytes appear to have specific CRF receptors. 4. In another experiment, both CRF and MSH antagonize tumor necrosis factor stimulation of immunocytes. Again, alpha-helical CRF antagonizes only CRF activity, further suggesting the presence of a separate CRF receptor on these cells.

Mytilus edulis, a marine bivalve mollusc, contains hemocytes that share certain properties with cells of the human monocyte-macrophage lineage. Because of this and previously reported similar responses to opioids between Mytilus hemocytes and human granulocytes, we determined whether Mytilus might also possess monokines or an immune monokine-like network. We chose to study two monokines, interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF). We found that Mytilus hemocytes respond to recombinant human IL-1 and TNF in a fashion similar to human granulocytes. Furthermore, we show that as in the human system, it appears that IL-1 mediates at least part of its effects through immunoreactive (ir) TNF in Mytilus. Finally, we show that Mytilus hemolymph contains ir IL-1 and TNF. To our knowledge, this is the first demonstration of these substances in this system.