Inducible nitric oxide synthase expression is increased in the alveolar compartment of asthmatic patients.

INTRODUCTION: Increased exhaled nitric oxide (NO) levels in asthma are suggested to be through inducible NO synthase (iNOS). The aim of this study was to investigate the expression of iNOS in bronchoalveolar lavage (BAL) cells and tissue from central and peripheral airways and compare it with the exhaled bronchial and alveolar NO levels in patients with asthma vs a control group. METHODS: Thirty-two patients with asthma (defined as controlled or uncontrolled according to Asthma Control Test score cut-off: 20) and eight healthy controls were included. Exhaled NO was measured, and alveolar concentration and bronchial flux were calculated. iNOS was measured in central and peripheral lung biopsies, as well as BAL cells. Bronchoalveolar lavage macrophages were stimulated in vitro, and iNOS expression and NO production were investigated. RESULTS: Expression of iNOS was increased in central airway tissue and the alveolar compartment in uncontrolled as compared to controlled asthmatics and healthy controls. There were no differences, however, in iNOS mRNA levels in total BAL cells in uncontrolled as compared to controlled asthma. Bronchoalveolar lavage cell mRNA levels of iNOS or iNOS expression in central and alveolar tissue did not relate to alveolar NO, nor to bronchial flux of NO. In vitro stimulation with leukotriene D4 increased iNOS mRNA levels and NO production in cultured BAL macrophages. CONCLUSION: The levels of both bronchial and alveolar iNOS are increased in uncontrolled as compared to controlled asthma. However, levels of iNOS in BAL macrophages were not reflected by alveolar NO. Both central and distal iNOS levels may reflect responsiveness to steroid treatment.

The confounding effect of the development of idiopathic orthostatic edema and thyrotoxcosis on weight fluctuation related to effects on free water clearance in a woman with long-standing surgically induced panhypopituitarism and diabetes insipidus.

PURPOSE: To evaluate the effect of idiopathic orthostatic edema and the effect of thyrotoxicosis on weight fluctuation and fluid retention in the presence of surgically induced panhypopituitarism and diabetes insipidus controlled with hormone replacement. MATERIALS AND METHODS: Dextroamphetamine sulfate was used for weight gain when no other etiologic factor was found. Methimazole was used when weight loss occurred when serum T4 and free T4 indicated thyrotoxicosis. RESULTS: Sympathomimetic amine therapy very effectively controlled the weight gain and methimazole controlled the weight loss. CONCLUSIONS: Hypopituitarism and diabetes insipidus controlled with hormone replacement do not protect against fluid retention from idiopathic edema.

Marked improvement of the autoimmune syndrome associated with autoimmune hepatitis by treatment with sympathomimetic amines.

PURPOSE: To evaluate the effect of sympathomimetic amine therapy for a life threatening autoimmune disorder. MATERIALS AND METHODS: Dextroamphetamine sulfate was used to treat edema, myalgia, and chronic fatigue associated with autoimmune hepatitis (AIH). RESULTS: Sympathomimetic amine therapy completely abrogated the symptoms associated with AIH. CONCLUSIONS: AIH should be added to the long list of chronic treatment-refractory conditions that respond quickly and effectively to treatment with sympathomimetic amines.

The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

Inducible nitric oxide synthase in tangle-bearing neurons of patients with Alzheimer's disease.

In Alzheimer's disease (AD), affected neurons accumulate beta amyloid protein, components of which can induce mouse microglia to express the high-output isoform of nitric oxide synthase (NOS2) in vitro. Products of NOS2 can be neurotoxic. In mice, NOS2 is normally suppressed by transforming growth factor beta 1 (TGF-beta 1). Expression of TGF-beta 1 is decreased in brains from AD patients, a situation that might be permissive for accumulation of NOS2. Accordingly, we investigated the expression of NOS2 in patients with AD, using three monospecific antibodies: a previously described polyclonal and two new monoclonal antibodies. Neurofibrillary tangle-bearing neurons and neuropil threads contained NOS2 in brains from each of 11 AD patients ranging in age from 47 to 81 years. NOS2 was undetectable in brains from 6 control subjects aged 23-72 years, but was expressed in small amounts in 3 control subjects aged 77-87 years. Thus, human neurons can express NOS2 in vivo. The high-output pathway of NO production may contribute to pathogenesis in AD.

Production of nitric oxide in the synovial membrane of rheumatoid and osteoarthritis patients.

We have demonstrated spontaneous nitric oxide (NO) production by primary synovial cultures from rheumatoid (RA) and osteoarthritis patients. Increased NO production followed addition of staphylococcal enterotoxin B. Immunochemical double staining with specific anti-human inducible NO synthase (iNOS) and nonspecific esterase (NSE), or anti-CD68 (markers for tissue macrophages) showed that although many lining layer cells in RA synovium expressed iNOS, most (approximately 90%) were NSE- and CD68-, with only a minor population (approximately 10%) which were iNOS+, CD68+/NSE+. These data demonstrate the capacity for high output of NO by human synovial tissue and show that, although human macrophages can express high levels of iNOS, the majority of cells expressing iNOS are fibroblasts. We also report that synoviocytes, and macrophage cell lines, cultured with the NO donor, S-nitroso-acetyl penicillamine, produced high concentrations of tumor necrosis factor (TNF)-alpha. These results suggest that NO may mediate pathology in RA through the induction of TNF-alpha production.

Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis.

The high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS;NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage. An average of 65% of the macrophages from 11 of 11 patients with untreated, culture-positive pulmonary tuberculosis reacted with an antibody documented herein to be monospecific for human NOS2. In contrast, a mean of 10% of bronchoalveolar lavage cells were positive from each of five clinically normal subjects. Tuberculosis patients' macrophages displayed diaphorase activity in the same proportion that they stained for NOS2, under assay conditions wherein the diaphorase reaction was strictly dependent on NOS2 expression. Bronchoalveolar lavage specimens also contained NOS2 mRNA. Thus, macrophages in the lungs of people with clinically active Mycobacterium tuberculosis infection often express catalytically competent NOS2.

Position of the greater trochanter and functional femoral antetorsion: Which factors matter in the management of femoral antetorsion disorders?

Aims: The primary aim of this study was to define and quantify three new measurements to indicate the position of the greater trochanter. Secondary aims were to define 'functional antetorsion' as it relates to abductor function in populations both with and without torsional abnormality. Patients and Methods: Three new measurements, functional antetorsion, posterior tilt, and posterior translation of the greater trochanter, were assessed from 61 CT scans of cadaveric femurs, and their reliability determined. These measurements and their relationships were also evaluated in three groups of patients: a control group (n = 22), a 'high-antetorsion' group (n = 22) and a 'low-antetorsion' group (n = 10). Results: In the cadaver group, the mean anatomical antetorsion was 14.7° (sd 8.5; 0 to 36.5) and the functional antetorsion 21.5° (sd 8.1; 3.6 to 44.3): the posterior tilt was 73.3° (sd 10.8; 46.9 to 88.7) and the posterior translation 0.59 (sd 0.2; 0.2 to 0.9). These measurements had excellent intra and interobserver agreement with a range from 0.93 to 0.99. When the anatomical antetorsion decreased, the greater trochanter was more tilted and translated posteriorly in relation to the axis of the femoral neck, and the difference between functional and anatomical antetorsion increased. The results the three patient groups were similar to those of the cadaver group. Conclusion: The position of the greater trochanter and functional antetorsion varied with anatomical antetorsion. In the surgical management of femoral retrotorsion, subtrochanteric osteotomy can result in an excessively posterior position of the greater trochanter and an increase in functional antetorsion. Cite this article: Bone Joint J 2018;100-B:712-19.

VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalized in articular cartilage during inflammatory arthritis.

The destruction of articular cartilage in immune inflammatory arthritic disease involves the proteolytic degradation of its extracellular matrix. The role of activated matrix metalloproteinases (MMPs) in the chondrodestructive process was studied by identifying a selective cleavage product of aggrecan in murine arthritis models initiated by immunization with either type II collagen or proteoglycan. We conducted semiquantitative immunocytochemical studies of VDIPEN341 using a monospecific polyclonal antibody requiring the free COOH group of the COOH-terminal Asn for epitope detection. This antibody recognizes the aggrecan G1 domain fragment generated by MMP [i.e., stromelysin (SLN) or gelatinase A] cleavage of aggrecan between Asn341-Phe342 but does not recognize intact aggrecan. VDIPEN was undetectable in normal mouse cartilage but was observed in the articular cartilage (AC) of mice with collagen-induced arthritis 10 d after immunization, without histological damage and clinical symptoms. This aggrecan neoepitope was colocalized with high levels of glycosaminoglycans (GAGs) in pericellular matrices of AC chondrocytes but was not seen at the articular surface at this early time. Digestion of normal (VDIPEN negative) mouse paw cryosections with SLN also produced heavy pericellular VDIPEN labeling. Computer-based image analysis showed that the amount of VDIPEN expression increased dramatically by 20 d (70% of the SLN maximum) and was correlated with GAG depletion. Both infiltration of inflammatory cells into the synovial cavity and early AC erosion were also very prominent at this time. Analysis of adjacent sections showed that both induction of VDIPEN and GAG depletion were strikingly codistributed within sites of articular cartilage damage. Similar results occurred in proteoglycan-induced arthritis, a more progressive and chronic model of inflammatory arthritis. These studies demonstrate for the first time the MMP-dependent catabolism of aggrecan at sites of chondrodestruction during inflammatory arthritis.

Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints.

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."

EDTA is superior to DTT treatment for overcoming the prozone effect in HLA antibody testing.

A limitation of solid-phase human leukocyte antigen (HLA) antibody assays is the falsely low/negative result of samples with high-titer antibodies, a phenomenon known as the prozone effect. Here we compared the efficacy of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect. A total of 21 serum samples were treated with either EDTA or DTT before HLA single antigen bead assay. The efficacy of prozone effect reversal, compared with untreated samples, was examined on fourfold, serially diluted samples, from neat to 1:256, using PBS as diluent. EDTA reversed the prozone effect in all tested samples, with an efficiency of greater than 84%, estimated by the ratio of undiluted sample mean fluorescence intensity (MFI) to peak MFI, for any given dilution. In contrast, the efficiency of DTT treatment was as low as 47%. These results show superior prozone effect reversal with EDTA treatment, compared with DTT.

The science of recalls.

Morbidity due to foodborne illnesses in the US has decreased over the last ten years. During the same time period recalls affecting the meat and poultry industry have increased from 38 in 1993 to a peak of 128 in 2002. Recalls due to L. monocytogenes (LM) and E. coli O157:H7 have accounted for the majority of recalls in recent years, while incidence rates for these pathogens have decreased. Incidence of Salmonella and E. coli O157:H7 cases since 1996 have decreased 17% and 42% respectively while product positives in ready to eat foods for LM has decreased from 3% in 1995 to 0.75% in 2003. In response to the increasing number of recalls, members of the meat and poultry industry have developed recall plans to effectively manage a recall crisis. A detailed recall plan which is tested through mock scenarios is essential to reducing the economic and negative consumer confidence impact of recalls.

GABAergic signalling in the immune system.

The GABAergic system is the main inhibitory neurotransmitter system in the central nervous system (CNS) of vertebrates. Signalling of the transmitter γ-aminobutyric acid (GABA) via GABA type A receptor channels or G-protein-coupled type B receptors is implicated in multiple CNS functions. Recent findings have implicated the GABAergic system in immune cell functions, inflammatory conditions and diseases in peripheral tissues. Interestingly, the specific effects may vary between immune cell types, with stage of activation and be altered by infectious agents. GABA/GABA-A receptor-mediated immunomodulatory functions have been unveiled in immune cells, being present in T lymphocytes and regulating the migration of Toxoplasma-infected dendritic cells. The GABAergic system may also play a role in the regulation of brain resident immune cells, the microglial cells. Activation of microglia appears to regulate the function of GABAergic neurotransmission in neighbouring neurones through changes induced by secretion of brain-derived neurotrophic factor. The neurotransmitter-driven immunomodulation is a new but rapidly growing field of science. Herein, we review the present knowledge of the GABA signalling in immune cells of the periphery and the CNS and raise questions for future research.

Mental stimulation increases circulating CD4-positive T lymphocytes: a preliminary study.

To stimulate the dorsolateral frontal cortex, 12 healthy, adult, human females played contract bridge for 1.5 h between initial and final blood sample collections. Flow cytometric analyses of samples, performed in triplicate, showed a significant increase in CD4-positive T lymphocytes. The dorsolateral frontal cortical thickness is significantly and bilaterally reduced in immune-incompetent female, nude mice. Thymic transplants reverse the deficient cortical thickness and CD4-positive cell numbers.

Platelet membrane early activation markers during prolonged storage.

The relationship between platelet aging and early markers of membrane activation have not been defined clearly. Activation markers expressed during prolonged storage are similar if not identical to those that appear after exposure to thrombin. Using flow cytometry, we investigated platelet membrane expression of CD62P, CD63, and annexin V binding (i.e., loss of membrane asymmetry) in platelets stored for up to 11 days under standard blood banking conditions. We compared five apheresis platelets to two random donor platelet concentrates, and to one pooled platelet preparation from six single platelet concentrates before and after exposure to thrombin. CD62P, CD63 expression, and annexin V binding increased during storage albeit with different kinetics. The differential increments observed between resting and thrombin (1 unit/ml) activated platelets showed an inverse correlation to storage time for CD62P, CD63, and annexin V binding, which was identical to published survival curves. A difference between apheresis platelets and platelet concentrates was observed only on day 1. Our data indicate that the in vitro platelet reserve activity to thrombin activation mirrors that of radiolabeled platelet survival in vivo and that platelet cross-sectional residual life span can explain their diminished capacity to respond to thrombin as a function of viability.

Complement levels in dogs with familial canine dermatomyositis.

CH50, C4, C2, and C3 levels were evaluated in 7 dogs affected with dermatomyositis and in 22 control dogs. Dogs with dermatomyositis did not have clinical evidence of active disease at the time of serum collection for complement assays. No absolute complement component deficiency was identified in dermatomyositis-affected dogs in this study; however, a statistical difference in C2 was identified between control dogs of non-collie breeds and control collies, suggesting there may be a breed difference in complement levels.

[Panorama ultrasonography of the abdominal wall for delineation of the anatomy and diagnosis of pathological findings]. / Panorama-Ultraschall der Bauchwand zur Darstellung der Anatomie und pathologischer Befunde.

PURPOSE: To assess extended field-of-view sonography for delineation of the anatomic structures of the abdominal wall and for the diagnosis of hernia. MATERIAL AND METHODS: In 34 cases (24 probands, 10 patients with abdominal wall defects) extended field-of-view sonography of the abdominal wall was performed with a 7.5 MHz transducer. Dynamic alignment of real-time images allows for depiction of regions of up to 60 cm in a single extended field-of-view image. A standardized axial image was obtained above and below the arcuate line and at the level of the defect, respectively. All images were evaluated by two blinded readers regarding the visibility of the anatomic structures and the delineation and extent of pathological changes. RESULTS: The abdominal wall was delineated with extended field-of-view sonography in all patients with a good image quality. The linea alba, the rectus muscle and the three lateral abdominal muscles, the rectus sheath, the peritoneum, and the subcutaneous fatty tissue were visible. In the patients with abdominal wall defects, the hernial sac with its contents, the extension of the defect, and the surrounding structures were clearly delineated. CONCLUSION: Extended field-of-view sonography allows for easily surveyed and reproducible documentation of the ultrasound examination of the abdominal wall. It is especially useful for the preoperative planning in patients with abdominal wall defects.

Pleural effusion secondary to thoracic metastatic mammary adenocarcinoma in a mare.

A 17-year-old Quarter Horse mare was examined nearly 3 years after excision and cryotherapy of a papillary mammary gland adenocarcinoma. The mare had been used for pleasure riding since surgery, but had recently developed progressive dyspnea. The mare had clinical evidence of pleural effusion, but died before further clinical examination and treatment were instituted. Necropsy revealed deep mammary masses with similar nodules in the deep inguinal, renal, and mediastinal lymph nodes and in the lungs, pericardium, visceral and parietal pleurae, and left ovary. The masses were identified as papillary mammary gland adenocarcinoma. Large volumes of free pleural and peritoneal fluid were detected. The pleural fluid contained similar neoplastic cells that could have been readily detected by exfoliative cytologic examination had the mare survived.

Common ventricle with separate pulmonary outflow chamber in a horse.

On the basis of clinical and laboratory examinations, a ventricular septal defect or a variant of the tetralogy of Fallot was suspected in a 3-year-old filly with a history of poor growth rate and exercise intolerance. The filly was euthanatized and found to have a 3-chambered heart (cor triloculare biatriatum). The heart had 2 normally formed atria and a large common ventricle into which the right and left atrioventricular orifices opened and from which the aorta arose. There was a small separate chamber from which the pulmonary trunk originated. This chamber communicated with the common ventricle through a large oval opening along the dorsal border of the displaced, interventricular septum.

Myelitis associated with protozoal infection in newborn calves.

Four newborn calves from different herds were examined because of recumbency and inability to rise. Abnormal physical findings were confined to the nervous and/or musculoskeletal systems. Because of poor prognosis, all calves were euthanatized and necropsied. The histopathologic findings were multifocal lymphocytic myelitis, meningitis, and encephalitis associated with protozoal cysts.