Limitations to intergenerational inheritance: subchronic paternal stress preconception does not influence offspring anxiety.

Independent studies have observed that a paternal history of stress or trauma is associated with his children having a greater likelihood of developing psychopathologies such as anxiety disorders. This father-to-child effect is reproduced in several mouse models of stress, which have been crucial in developing a greater understanding of intergenerational epigenetic inheritance. We previously reported that treatment of C57Bl/6J male breeders with low-dose corticosterone (CORT) for 28 days prior to mating yielded increased anxiety-related behaviours in their male F1 offspring. The present study aimed to determine whether subchronic 7-day CORT treatment of male mice just prior to mating would be sufficient to induce intergenerational modifications of anxiety-related behaviours in offspring. We report that subchronic CORT treatment of male breeders reduced their week-on-week body weight gain and altered NR3C1 and CRH gene expression in the hypothalamus. There were no effects on sperm count and glucocorticoid receptor protein levels within the epididymal tissue of male breeders. Regarding the F1 offspring, screening for anxiety-related behaviours using the elevated-plus maze, light-dark box, and novelty-suppressed feeding test revealed no differences between the offspring of CORT-treated breeders compared to controls. Thus, it is crucial that future studies take into consideration the duration of exposure when assessing the intergenerational impacts of paternal health.

Exercise alters mouse sperm small noncoding RNAs and induces a transgenerational modification of male offspring conditioned fear and anxiety.

There is growing evidence that the preconceptual lifestyle and other environmental exposures of a father can significantly alter the physiological and behavioral phenotypes of their children. We and others have shown that paternal preconception stress, regardless of whether the stress was experienced during early-life or adulthood, results in offspring with altered anxiety and depression-related behaviors, attributed to hypothalamic-pituitary-adrenal axis dysregulation. The transgenerational response to paternal preconceptual stress is believed to be mediated by sperm-borne small noncoding RNAs, specifically microRNAs. As physical activity confers physical and mental health benefits for the individual, we used a model of voluntary wheel-running and investigated the transgenerational response to paternal exercise. We found that male offspring of runners had suppressed reinstatement of juvenile fear memory, and reduced anxiety in the light-dark apparatus during adulthood. No changes in these affective behaviors were observed in female offspring. We were surprised to find that running had a limited impact on sperm-borne microRNAs. The levels of three unique microRNAs (miR-19b, miR-455 and miR-133a) were found to be altered in the sperm of runners. In addition, we discovered that the levels of two species of tRNA-derived RNAs (tDRs)-tRNA-Gly and tRNA-Pro-were also altered by running. Taken together, we believe this is the first evidence that paternal exercise is associated with an anxiolytic behavioral phenotype of male offspring and altered levels of small noncoding RNAs in sperm. These small noncoding RNAs are known to have an impact on post-transcriptional gene regulation and can thus change the developmental trajectory of offspring brains and associated affective behaviors.

Elevated paternal glucocorticoid exposure alters the small noncoding RNA profile in sperm and modifies anxiety and depressive phenotypes in the offspring.

Recent studies have suggested that physiological and behavioral traits may be transgenerationally inherited through the paternal lineage, possibly via non-genomic signals derived from the sperm. To investigate how paternal stress might influence offspring behavioral phenotypes, a model of hypothalamic-pituitary-adrenal (HPA) axis dysregulation was used. Male breeders were administered water supplemented with corticosterone (CORT) for 4 weeks before mating with untreated female mice. Female, but not male, F1 offspring of CORT-treated fathers displayed altered fear extinction at 2 weeks of age. Only male F1 offspring exhibited altered patterns of ultrasonic vocalization at postnatal day 3 and, as adults, showed decreased time in open on the elevated-plus maze and time in light on the light-dark apparatus, suggesting a hyperanxiety-like behavioral phenotype due to paternal CORT treatment. Interestingly, expression of the paternally imprinted gene Igf2 was increased in the hippocampus of F1 male offspring but downregulated in female offspring. Male and female F2 offspring displayed increased time spent in the open arm of the elevated-plus maze, suggesting lower levels of anxiety compared with control animals. Only male F2 offspring showed increased immobility time on the forced-swim test and increased latency to feed on the novelty-supressed feeding test, suggesting a depression-like phenotype in these animals. Collectively, these data provide evidence that paternal CORT treatment alters anxiety and depression-related behaviors across multiple generations. Analysis of the small RNA profile in sperm from CORT-treated males revealed marked effects on the expression of small noncoding RNAs. Sperm from CORT-treated males contained elevated levels of three microRNAs, miR-98, miR-144 and miR-190b, which are predicted to interact with multiple growth factors, including Igf2 and Bdnf. Sustained elevation of glucocorticoids is therefore involved in the transmission of paternal stress-induced traits across generations in a process involving small noncoding RNA signals transmitted by the male germline.

Recurrence of light chain nephropathy in a renal allograft. A case report and review of the literature.

Myeloma and monoclonal gammopathies can affect the kidney in many ways with cast nephropathy and light chain deposition disease being the most commonly recognised histological entities. Renal transplantation in these patients remains controversial both because of the risk of recurrent disease affecting the graft and also because of concerns around the possibility of disease relapse within the patient. We suggest that the histological pattern of disease within the native kidneys is crucial in the overall assessment of these patients for renal transplantation. Those patients in whom renal deposition of light chains is associated with a proliferative glomerulonephritis have a considerably worse graft survival than those presenting with cast nephropathy.

Small intestinal infarction: a fatal complication of systemic oxalosis.

Primary hyperoxaluria is a rare genetic disorder characterised by calcium oxalate nephrolithiasis and nephrocalcinosis leading to renal failure, often with extra-renal oxalate deposition (systemic oxalosis). Although ischaemic complications of crystal deposition in vessel walls are well recognised clinically, these usually take the form of peripheral limb or cutaneous ischaemia. This paper documents the first reported case of fatal intestinal infarction in a 49 year old woman with systemic oxalosis and advocates its consideration in the differential diagnosis of an acute abdomen in such patients.

Studies of epitope restriction on myeloperoxidase (MPO), an important antigen in systemic vasculitis.

Anti-neutrophil cytoplasmic antibodies are important components of the inflammatory response in patients with systemic vasculitis. Their role in the pathogenesis of these conditions remains incompletely defined. Several antigens have been identified, and MPO is one of the most important. To gain more understanding of the immune mechanisms involved, we were keen to see if the antibody response to MPO was restricted, or whether there was a general loss of tolerance to the whole surface of the molecule. To study the epitopes we employed both ELISA and biosensor technology, and were able to demonstrate restriction both in the number and localization of the epitopes being recognized.

Antigen specificity in hydralazine associated ANCA positive systemic vasculitis.

The anti-hypertensive agent hydralazine can cause a lupus-like syndrome characterized by serosal inflammation, arthralgias and rashes. The kidneys however are usually spared. The condition is characterized by circulating immune complexes and antinuclear antibodies, whilst antibodies against double-stranded DNA are rare. Hydralazine can also cause a systemic vasculitis with a pauci-immune rapidly progressive glomerulonephritis, which is associated with autoantibodies directed against components of the neutrophil cytoplasm. In this study, ten patients with hydralazine-induced vasculitis had antibodies with specificities for both myeloperoxidase and lactoferrin. We suggest that this particular pattern of autoantibodies, together with antibodies with reactivity against nuclear components including double-stranded DNA, are characteristic findings in hydralazine-induced vasculitis. In addition, renal involvement appears to be more common in this group of patients with vasculitis than in those with the lupus-like syndrome.

Neutrophil and recombinant myeloperoxidase as antigens in ANCA positive systemic vasculitis.

Myeloperoxidase (MPO) is one of the major autoantigens recognized by anti-neutrophil cytoplasm antibodies. The association of this antigen with specific disease entities requires that there is a source of pure antigen present in large quantities. Further delineation of the molecular mechanisms involved in the antigen-antibody interaction requires the ability to manipulate the molecule. The expression of recombinant MPO in Chinese hamster ovary cells has produced a source of pure protein, suitable for molecular studies. We have shown that this protein is an antigen recognized by 95% of anti-MPO antibodies from patients with systemic vasculitis. This recombinant molecule will be of use in providing an additional specific solid-phase assay for these antibodies and further forms of this protein which mirror the antigenicity of native MPO more exactly may replace chemically purified antigen. It will also be of great value in studies examining the epitopes recognized by anti-MPO antibodies and in studies of immunoregulation and T cell activation.

Anti-neutrophil cytoplasm antibodies and anti-glomerular basement membrane antibodies: two coexisting distinct autoreactivities detectable in patients with rapidly progressive glomerulonephritis.

Circulating autoantibodies against the Goodpasture antigen (alpha 3 chain of type IV collagen) in the glomerular basement membrane (anti-GBM) and anti-neutrophil cytoplasm antibodies (ANCA) are each associated clinically with the development of a rapidly progressive glomerulonephritis. Antibodies with both these specificities coexist in a subset of patients, raising the possibility that they might be a result of cross-reactivity. In this study we have shown that 21% of patients with anti-GBM antibodies also had ANCA, and by using cross-inhibition assays, antigen-specific enzyme-linked immunosorbent assays, and Western blot analysis, these were shown to be two distinct populations of autoantibodies. In patients with both specificities, a greater proportion of the ANCA had specificity for myeloperoxidase (73.5%) than in patients with ANCA alone (36.6%). The presence of ANCA should be ascertained in all patients with anti-GBM disease as the prognosis for these double-positive patients may be dependent on both populations of antibodies.

Antineutrophil cytoplasm autoantibodies and vasculitis.

Antineutrophil cytoplasm autoantibodies are useful diagnostic serologic markers for a variety of well-known primary vasculitic syndromes, including Wegener's granulomatosis, microscopic polyangiitis, and idiopathic necrotizing and crescentic glomerulo-nephritis. More recently antineutrophil cytoplasm autoantibodies have been found in other vasculitic syndromes, such as Churg-Strauss syndrome, Henoch-Schönlein purpura, and some nonvasculitic diseases such as rheumatoid arthritis, inflammatory bowel disease, and autoimmune hepatobiliary diseases. There is now evidence to suggest that infection might be an important etiologic factor in the development of antineutrophil cytoplasm autoantibody-associated vasculitides. This link has been strengthened by in vitro data that suggest that antineutrophil cytoplasm autoantibodies are directly involved in the pathogenesis of antineutrophil cytoplasm autoantibody-associated vasculitides.

Autoantibodies to lactoferrin and histone in systemic vasculitis identified by anti-myeloperoxidase solid phase assays.

We aimed at confirming the antigen specificity recognized by anti-neutrophil cytoplasm antibodies (ANCA) in patients presenting systemic vasculitis with anti-myeloperoxidase (MPO) activity on ELISA. Thirty-five consecutive patients with reactivity in anti-MPO ELISA and systemic microscopic vasculitides were tested in slot and Western blot analyses. Eleven of 35 sera exhibited binding in Western blot studies with the MPO preparation used in the ELISA: five sera bound at the size of MPO, but five sera reacted with a 78 kD species (p78) co-purifying with MPO, and one serum blotted both MPO and p78. Sequence analysis and antigen-specific assays including Western blot studies showed that p78 is lactoferrin. All anti-lactoferrin positive sera, but no anti-MPO positive sera, also exhibited anti-nuclear binding on HEp2 cells with specificity for histone. We concluded that: (a) a subgroup of patients presenting systemic vasculitis with false anti-MPO reactivity on ELISA had anti-lactoferrin antibodies; (b) anti-lactoferrin was associated with anti-nuclear activity with specificity for histone; (c) these patients had systemic vasculitis without histological evidence of immune complex deposition.

Does truly 'idiopathic' crescentic glomerulonephritis exist?

Crescentic glomerulonephritis is usually classified into antiglomerular basement membrane (GBM) disease, immune-complex disease, or pauci-immune crescentic nephritis. The last category includes patients with systemic vasculitis as well as 'idiopathic' isolated crescentic nephritis. The presence of anti-neutrophil cytoplasmic antibodies (ANCA) in many patients with apparently isolated crescentic nephritis suggests that this represents a renal-limited form of vasculitis, and that truly 'idiopathic' crescentic nephritis is a very rare entity. We reviewed all renal biopsies with extracapillary proliferation seen at our centre since the availability of an ANCA assay (4-year period). There were 89 such biopsies of a total of 1240, of which 82 had sufficient details for further analysis. Of these, 10 had anti-GBM disease, 35 had epithelial proliferation associated with a variety of other diseases, and 36 had ANCA-associated disease. Nine of this last group had no extrarenal features and would previously have been classified as 'idiopathic' crescentic glomerulonephritis. The single remaining patient had an inactive glomerulonephritis with a scarred crescent; the predominant lesion was an interstitial nephritis. We therefore conclude that truly 'idiopathic' crescentic nephritis is very rare, if it exists at all. The ability to provide a practically complete classification of crescentic nephritis has important prognostic and therapeutic consequences.

Lactoferrin co-purifies with myeloperoxidase and is recognised by anti-neutrophil cytoplasm antibodies.

Thirty-five sera with binding greater than 20% in a myeloperoxidase (MPO, Calbiochem) ELISA were tested in Western blot analyses. 5/35 blotted MPO, but 5/35 blotted lactoferrin (LF) contaminating the commercial MPO preparation. All the anti-LF positive sera, but none of the anti-MPO positive sera, also exhibited anti-nuclear binding on Hep2 cells. Three of the patients with anti-LF antibodies had vasculitis affecting areas additional to the pulmonary-renal involvement which characterised the patients with anti-MPO antibodies.

ANCA and anti-GBM antibodies in RPGN.

ANCA and anti-GBM antibodies can occur together in RPGN. These patients are rare and have two distinct populations of antibodies and the ANCA specificity tends to be for myeloperoxidase more often than expected.

Thermal dye measurements of extravascular lung water in critically ill patients. Intravascular Starling forces and extravascular lung water in the adult respiratory distress syndrome.

To assess the concurrent influence on extravascular lung water (EVLW) content of the intravascular Starling forces, the pulmonary capillary wedge pressure (PCWP), and the colloid osmotic pressure (COP), we measured EVLW by the thermal green dye technique in 174 patients with and without radiographically defined pulmonary edema; in the former group, patients with cardiac (CPE) and noncardiac (NCPE) causes of pulmonary edema were compared (study A). In 119 patients, EVLW was again measured one to three days later (study B). Patients with CPE demonstrated a significantly lower EVLW (9.3 +/- 3.9 ml/kg) (mean +/- SD) than patients with NCPE (14.5 +/- 4.9 ml/kg; p less than 0.05), despite a higher mean PCWP in the former group (20 +/- 7 mm Hg) than in the latter (12 +/- 6 mm Hg; p less than 0.05). In patients potentially with only a hydrostatic cause of pulmonary edema in study A, regression analysis demonstrated the following: EVLW = 3.2 + 0.30 PCWP (r2 = 0.38; p less than 0.005); and in patients with NCPE, EVLW = 10.9 + 0.304 PCWP (r2 = 0.17; p less than 0.01). In study B the change (delta) in EVLW between the two studies was described as follows: delta EVLW = 0.25 + 0.173 delta PCWP (p less than 0.01) + 0.663 group NCPE (p, not significant) + 0.236 group NCPE X delta PCWP (p less than 0.01). This latter equation indicated that the EVLW content manifested a greater change with concurrent alterations in the PCWP in patients with NCPE than was found in patients with only a hydrostatic influence to EVLW formation. Therefore, NCPE is characterized by a greater measurable thermal green dye EVLW than is observed in CPE at any given PCWP, and the PCWP synergistically influences EVLW accumulation in both CPE and NCPE.

Clinical studies of measuring extravascular lung water by the thermal dye technique in critically ill patients.

We measured extravascular lung water (EVLW) by the thermal-dye technique in a broad group of critically ill patients who had either acute cardiac or noncardiac illnesses. A portable AP supine chest roentgenogram, reviewed blindly, was used to classify patients as to the presence or absence of pulmonary edema; by clinical history we categorized patients into either a cardiac or noncardiac (ie, ARDS) group. With a normal chest roentgenogram, the mean EVLW was 5.6 +/- 1.8 ml/kg, and the pulmonary capillary wedge pressure (PCWP) was 11.3 +/- 5.3 mm Hg (mean +/- SD). In contrast, patients with pulmonary edema on a cardiac basis had a mean EVLW of 10.2 +/- 3.1 ml/kg (mean PCWP, 20.5 +/- 8.2 mm Hg), while patients with clinically defined noncardiac pulmonary edema and a normal PCWP (11.6 +/- 5.7 mm Hg) had a mean EVLW of 15.8 +/- 4.6 ml/kg, significantly higher than in the cardiac group (p less than 0.001). On a severity system of 014, the EVLW increased in parallel to the severity of the chest radiologic appearance of edema in both the cardiac (r2 = .44; p less than 0.001) and noncardiac (r2 = .59; p less than 0.001) patients. This study defined a normal range of thermal-dye EVLW in critically ill patients without radiologic evidence of pulmonary edema. We further demonstrated the increased pulmonary microvascular permeability of noncardiac pulmonary edema compared with cardiac edema by the greater EVLW at normal microvascular hydrostatic pressures in the former group.

beta-Naphthylamidase activity of the cell surface of Ehrlich ascites cells. Reversible control of enzyme activity by metal ions and thiols.

Ehrlich ascites tumour cells grown in mice have a cell-surface trypsin-like neutral protease (TLNP) which is not inhibited by high-mol.-wt inhibitors of trypsin. This enzyme is inhibited by low concentrations of zinc, which may be removed by chelating agents, with the consequent return of enzymic activity. Gold, provided as the drugs aurothiomalate or auranofin, also inhibits TLNP. The gold can be removed from the enzyme by incremental addition of thiols. The mechanisms of gold transfer to the active site to cause inhibition and subsequent removal of gold with reactivation of TLNP, have been shown to be controlled by reversible thiol-exchange reactions.