Origin of oestrus-associated glycoproteins in bovine oviductal fluid.

The aim of the present study was to determine whether the synthesis of an oestrus-associated protein found in bovine oviductal fluid varies with oviductal region, stage of cycle or day of pregnancy. Explant culture was performed using oviducts recovered from naturally cycling animals either at oestrus or 12-14 days after oestrus. Three oviductal regions, the preampulla, ampulla and isthmus, were cultured individually in the presence of 20 muCi [35S]methionine in serum-free medium for 6 h at 37 degrees C. Synthesis of oestrus-associated protein was assessed by one-dimensional SDS-PAGE, fluorography and densitometry of radiolabelled bands. Significantly more oestrus-associated protein was synthesized by the ampullar region of the oviduct, although it was detected in explant culture media from both the isthmic and preampullar regions. A polyclonal antibody produced against oestrus-associated protein was used to localize the protein in paraffin-embedded sections of oviductal explant cultures and other bovine tissues. Localization of the protein in oviductal tissue sections varied with stage of cycle (oestrus > luteal > pregnant) and region of oviduct (ampulla > preampulla/isthmus). These findings indicate an effect of oviductal region and hormonal state (cycling versus pregnant) on the synthesis and secretion of the oestrus-associated protein. Lectin affinity studies indicated that galactosyl(beta 1,3)N-acetylgalactosamine and N-acetylglucosamine residues are associated with the oestrus-associated protein.


Factors from fluid of the ovarian pocket that stimulate sperm motility in domestic hens.

Fluid was collected from the region of the peritoneum surrounding the ovarian pocket of domestic hens at about the time of ovulation. This fluid, diluted to 10%, increased the motility of chicken spermatozoa by a maximum of sixfold at 40 degrees C in vitro. Gel filtration revealed two peaks of motility-stimulating activity: one was identified as calcium and the other as a heat-labile substance of low M(r) (200). It is suggested that this motility-stimulating activity may facilitate fertilization, as spermatozoa are normally stored in a quiescent state within the oviduct and can be passively transported by cilia to the site of fertilization in the ovarian pocket.


Expression of mRNA for follicle-stimulating hormone suppressing protein in ovarian tissues of cows.

The expression of bovine follicle-stimulating hormone (FSH)-suppressing protein (FSP) mRNA was investigated in different ovarian tissues of cows. Northern blot analysis, using a cDNA probe to bovine FSP, demonstrated that the FSP gene in the bovine ovary is highly expressed in a pool of isolated granulosa cells. Two bands (2.8 and 1.8 kb) were observed in all tissues expressing the mRNA. FSP mRNA was low in small antral follicles and increased in growing follicles to reach a maximum in preovulatory follicles. Low amounts of mRNA of steady state FSP were observed in all stages of the corpus luteum as well as in the corpus luteum of pregnant cows, in the corpus albicans and theca tissue, whereas this mRNA could not be detected in the liver. These results are consistent with the hypothesis that, in cows, FSP functions as an autocrine regulator in developing follicles to facilitate luteinization of granulosa cells.


Plasma melatonin profiles of Romney Marsh sheep in natural photoperiod and in acutely extended darkness.

Plasma melatonin was measured at the summer and winter solstices and the autumn and spring equinoxes in Romney Marsh sheep held under natural conditions in South Australia (35 degrees S). The amount of melatonin detected was generally related to the extent of natural darkness, though the melatonin onset was particularly delayed after dusk in winter compared with other seasons. The duration of detectable melatonin was shorter in summer than at any other season. After each initial 24 h sampling, the sheep were resampled for a further 24 h in acutely extended darkness to mark the phase and duration of suprachiasmatic nuclei activity which is believed to be the source of the melatonin signal. The onset of high plasma melatonin was earlier than the time of natural sunset in spring and summer, but not different from the time of natural sunset in autumn and winter. The offset of high plasma melatonin was later than the time of natural sunrise at all times of year and particularly so in summer. Under the extended dark conditions, the duration of detectable melatonin was longer than that under natural photoperiod at all seasons of the year and the duration of melatonin was again shorter in summer than winter. If melatonin measurements under the conditions of extended darkness do reflect the phase and duration of suprachiasmatic nuclei function then the natural light of the photoperiod can, particularly during long photoperiod conditions, mask the expression of the pacemaker. The findings may have implications for the timing of the breeding season in Romney Marsh sheep.


Observations on the turkey oviductal sperm-storage tubule using differential interference contrast microscopy.

Squash preparations of unfixed, uterovaginal junction mucosae revealed that openings to sperm-storage tubules were round or slit-like and were surrounded by either cilia, which were part of the uterovaginal junction surface epithelium, or nonciliated cells resembling the sperm-storage tubule epithelium. By focusing on different levels of the sperm-storage tubule (optical sectioning), connective tissue fibres and cells between individual sperm-storage tubules, epithelium and lumen of sperm-storage tubules containing resident spermatozoa were observed. An optical section through the sperm-storage tubule epithelium revealed basal nuclei and associated nucleoli, and refractile supranuclear lipid droplets. Luminal spermatozoa were distributed primarily in the distal third of the sperm-storage tubule and nearly always formed a tight bundle at its base. These spermatozoa were often observed slowly and synchronously oscillating. In two-thirds of the 30-week-old, non-photostimulated hens, sperm-storage tubules were fully formed. In contrast, the remaining hens possessed bud-like surface invaginations lacking discernible lumina. It was concluded that differential interference contrast microscopy offers better spatial and optical resolution of the sperm-storage tubule than other modes of light microscopy.


Effect of pregnant mares' serum gonadotrophin on the micromorphology of antral follicles in heifers, with special reference to atypical granulosa cells.

Five Dutch-Friesian heifers were injected i.m. with 3000 iu pregnant mares' serum gonadotrophin (PMSG) on day 10 of the oestrous cycle, to study the effects on the number and micromorphological quality of antral follicles (> or = 0.3 mm in diameter). The ovaries were collected 48 h after PMSG injection. As well as the presence of mitotic figures and the absence of pyknotic nuclei in the granulosa, atypical granulosa cells were found in nonatretic follicles. These cells had an oblong nucleus and stained with toluidine blue. They were characterized by their dark cell matrix, and the presence of numerous free ribosomes and intermediate filaments of varying quantity. Atypical granulosa cells were micromorphologically similar to fibroblast-like cells in the theca. Their presence coincided with the occurrence of degenerative changes in the cytoplasm of nearby granulosa cells and they were more frequent in atretic follicles. The presence of atypical granulosa cells in follicles hitherto called nonatretic is therefore probably associated with the onset of follicular atresia. In the PMSG-treated heifers, the mean number of large (> or = 6.0 mm in diameter) antral follicles was greater than in the control group (18.4 +/- 4.0 versus 3.0 +/- 1.0), because of an increase in the number of large nonatretic follicles (11.8 +/- 4.4 versus 0.4 +/- 0.2). After hormone treatment, the mean number of medium-sized (3.0-5.9 mm) nonatretic follicles also increased (6.4 +/- 1.3 versus 1.8 +/- 1.0). PMSG did not change the mean number of nonatretic follicles < 3.0 mm or that of atretic follicles in the different size categories. However, when follicles hitherto called nonatretic, with atypical granulosa cells, were taken together with the group of atretic follicles, PMSG appeared to increase the mean number of large atretic follicles (13.6 +/- 2.4 versus 3.0 +/- 1.0). The mean number of medium-sized and large nonatretic follicles without atypical granulosa cells was markedly increased (3.8 +/- 1.0 versus 0.2 +/- 0.2 and 4.6 +/- 1.9 versus 0.0, respectively). The data demonstrate that PMSG stimulates the formation not only of nonatretic follicles > or = 3.0 mm, but also of atretic follicles > or = 6.0 mm.


Superoxide dismutase activity, lipid peroxide production and corpus luteum steroidogenesis during natural luteolysis and regression induced by oestradiol deprivation of the ovary in pseudopregnant rabbits.

The relationship of oxygen free radicals to corpus luteum function in rabbits was explored during various stages of pseudopregnancy, including natural and induced luteal regression. Induced luteolysis was achieved during mid-pseudopregnancy by removal of an oestradiol capsule placed at the onset of pseudopregnancy, which suppressed ovarian oestradiol production. Activity of manganese superoxide dismutase (Mn SOD) was significantly and positively correlated with ovarian progesterone production (P < 0.01) throughout pseudopregnancy and during natural regression. Oestradiol deprivation for 12, 24 or 72 h resulted in declines in Mn SOD activity and progesterone secretion, although Mn SOD rose and corpus luteum steroidogenesis was restored to normal when the capsule was replaced for 48 h before assessment, having been removed for 24 h. Lipid peroxide and progesterone concentrations were not correlated, although a significant rise in lipid peroxides in the luteal tissue was detected after deprivation of oestradiol for 72 h. Changes in progesterone production and Mn SOD activity were not associated with alterations in concentration of prostaglandin F metabolite. These data suggest that Mn SOD may be involved in regulating function of the corpus luteum during pseudopregnancy in rabbits and that oxygen free radicals may play a role in regression of corpus luteum in this species.


Seasonal pattern of luteinizing hormone and testosterone pulsatile secretion in young adult red deer stags (Cervus elaphus) and its association with the antler cycle.

Blood from stages aged 15 months (n = 6) was sampled at monthly intervals every 30 min for 24 h for 12 months, at 45 degrees S in New Zealand. Three extra samplings each for 24 h were carried out at about the anticipated time of antler casting. All samples were analysed for luteinizing hormone (LH) and testosterone and the resulting data further analysed by the Pulsar pulse detection routine. The animals were kept indoors under natural daylength and were fed ad libitum. All animals were weighed, antler status and size recorded and testes diameter was measured on each sampling day. Mean LH and testosterone pulsatily and plasma concentration varied seasonally. LH pulse frequency was low during autumn (2.5 pulses in 24 h), winter (1.0-1.5 pulses in 24 h) and early spring (1 pulse in 24 h) and lowest in late spring (0.2 pulse in 24 h) before rising in summer (1.0-4.0 pulses in 24 h). LH pulse amplitude and mean plasma concentration were low (< 1 ng ml-1) from March to November (autumn-spring); both rose to a peak in January (summer) of 3.4 and 1.6 ng ml-1, respectively. Testosterone pulse frequency was generally similar to LH except that slightly more pulses of testosterone than of LH were detected from March to November and more pulses of LH from November to February (summer). Testosterone pulse amplitude fell from March to November (5.3 ng ml-1 to undetectable) although there was a conspicuous peak in July (midwinter) of almost 5 ng ml-1.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of testosterone and bovine follicular fluid on concentrations of luteinizing hormone and follicle-stimulating hormone in plasma of castrated rams that are homozygous carriers or non-carriers of the Booroola fecundity gene.

Castrated adult FecBFecB and Fec+Fec+ Booroola rams were injected with charcoal-treated bovine follicular fluid (bFF) (a source of inhibin-like activity) or given testosterone implants to examine whether the fecundity gene (FecB) influences sensitivity to negative feedback hormones in males. Mean concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) did not differ between genotypes before treatment. In Expt 1, injections of 5 ml bFF, but not of 1 ml (each given four times at intervals of 8 h), significantly (P < 0.05) depressed concentrations of LH and FSH, but there was no effect of genotype. After treatment, gonadotrophin concentrations returned to pretreatment values and for 2-2.5 days scaled (divided by pretreatment mean) LH values (235 +/- 49 for FecBFecB and 96 +/- 26% for Fec+Fec+ rams; P < 0.05) and scaled FSH values (106 +/- 5 for FecBFecB and 85 +/- 5% for Fec+Fec+ rams; P < 0.05) were significantly higher in FecBFecB than in Fec+Fec+ rams in the group that received 5 ml bFF. Irrespective of genotype, treatment with 5 ml bFF did not reduce mean FSH to concentrations observed in testis-intact rams. In Expt 2, Silastic envelopes were implanted subdermally to give physiological or supraphysiological circulating concentrations of testosterone. Both doses significantly reduced scaled LH values in a biphasic manner, such that there was an initial suppression followed by a short-lived increase. During the initial period of suppression in the lower dose group, mean scaled LH values were significantly higher in FecBFecB than in Fec+Fec+ rams (48.3 +/- 7.5 versus 23.1 +/- 5.5%; P < 0.05). Low doses of testosterone decreased LH pulse frequency in both genotypes but decreased (P < 0.05) pulse amplitude and mean concentrations in the Fec+Fec+ animals only. In nonimplanted control rams, mean LH concentrations (in samples taken every 10 min for 12 h) were significantly lower in FecBFecB than in Fec+Fec+ rams (0.6 +/- 0.2 versus 1.3 +/- 0.1 ng ml-1; P < 0.05). The mean FSH response to testosterone was not related to genotype. These data suggest that expression of the FecB gene results in an altered sensitivity of the pituitary gland to changes in negative feedback from testicular hormones and that, irrespective of genotype, neither testosterone nor inhibin-like activity alone can fully control FSH secretion in castrated rams.


Influence of prenatal photoperiod on postnatal prolactin secretion in red deer (Cervus elaphus).

Plasma prolactin concentrations were higher (P < 0.001) in newborn red deer calves whose mothers had been maintained for the last 14 weeks of gestation in long days (18 h light) (group L, n = 9) than in those whose mothers had been kept over the same period in short days (6 h light) (group S, n = 5). After transfer of all hinds and suckled calves on the day of birth to constant intermediate daylength (12 h light), prolactin concentrations decreased exponentially (P < 0.001) in group L calves, but not in group S, during the first 21 days. Thereafter, prolactin fell to a nadir in group L calves and rose to peak values in group S calves at 8-12 weeks post partum (P = 0.003), before converging again by 14 weeks. The pattern of prolactin secretion over the first 14 weeks of life was therefore significantly affected by prenatal photoperiod. Plasma prolactin concentrations in the adult hinds were higher (P < 0.001) in group L than group S at 4-10 weeks before parturition; they were similarly high around parturition and fell thereafter to baseline values after 7 weeks. These results provide evidence that deer fetuses respond to photoperiodic information, thereby acquiring a photoperiodic history in utero that influences postnatal responses to photoperiod.


The relation of extraarticular tenderness to inflammatory joint disease and personality in patients with rheumatoid arthritis.

We measured the pain tenderness threshold at 16 fibrositic tender points in 44 consecutive patients with rheumatoid arthritis (RA) attending the outpatient rheumatology clinic of a university hospital. Pressure threshold measurements were transformed to z units to equalize the weights of the values at different anatomic sites and were then summed. This pain tenderness score correlated with the joint score index (p less than 0.02, r = -0.363), signifying a low pain threshold in the patients with a high joint score index. In contrast to this, the pain tenderness score was not explained by either personality factors or the generalized disease activity measuring variables (erythrocyte sedimentation rate, C-reactive protein). Our results show that the fibrositic point tenderness is real in RA, and that the tenderness is augmented near the active joints. The pain tenderness score of patients with RA is not affected by the subject's personality but may relate to sensitization of the pain fibers in arthritic joints.


Calprotectin in patients with rheumatoid arthritis: relation to clinical and laboratory variables of disease activity.

Calprotectin (L1) is a major granulocyte and monocyte protein which is released during activation of these cells. The plasma level of L1 is thought to reflect disease activity in rheumatoid arthritis (RA). In our cross sectional study of 70 patients with RA, L1 had significant correlations with erythrocyte sedimentation rate (r = 0.50), C-reactive protein (r = 0.58), orosomucoid (r = 0.62), platelet count (r = 0.42), leukocyte count (r = 0.33) and IgM rheumatoid factor (r = 0.32); and with the following clinical variables: number of swollen joints (r = 0.24), grip strength (r = -0.22), PIP joint circumferences (r = 0.33) and a combined global assessment score (r = 0.24). L1 was higher in seropositive (median 14,861 micrograms/l) than seronegative patients (median 10,487 micrograms/l) (p less than 0.03).


Accelerated nodulosis during low dose methotrexate therapy for rheumatoid arthritis. An analysis of ten cases.

To obtain information on the occurrence of accelerated nodulosis during methotrexate (MTX) for rheumatoid arthritis (RA), localization, size and presence in heart and lungs of these nodules, predisposing factors, relationship with other extraarticular manifestations (EAM) and their histological features. Ten patients with accelerated nodulosis were studied. Four participated in a double blind study of MTX and azathioprine. Patient characteristics, localization, size and histopathology of new nodules were determined. Echocardiography and chest roentgenograms were performed. Accelerated nodulosis occurred exclusively during treatment with MTX in our double blind study. The estimated incidence was 8%. One patient reported was rheumatoid factor negative. Newly developed nodules were small, and located in the fingers in all patients and in additional sites in 7. Pretreatment nodules were not found in the fingers. In one patient nodules on the mitral valve were found, but this was not likely to be associated with the use of MTX. No new pulmonary nodules were found. Other EAM developed concurrently in 4 patients. Histopathology revealed typical rheumatoid nodules. In 3 patients nodulosis regressed after MTX was stopped. In 2 of them they recurred after a rechallenge. Accelerated nodulosis during MTX for RA is not rare, and occurs despite good clinical response of polyarthritis. Rheumatoid factor positivity is not a prerequisite. New nodules are small and preferentially located in the fingers. Recurrence after rechallenge with MTX suggests causality.


Psoriatic arthritis: clinical response and side effects to methotrexate therapy.

In the last decade, methotrexate (MTX) has emerged as a useful second line agent for a variety of arthritides. However, there still exists some reluctance for its wider use mainly because of concerns about its liver side effects. We describe our clinical experience with this drug in psoriatic arthritis (PsA). The study group included 24 men and 16 women, with a mean age of 47 years (16-75), with oligoarticular (13) or polyarticular (27) involvement, with a mean disease duration of 12 years (1-36). Patients received a mean dose of 11.2 mg of MTX orally/week during a mean period of 34 months (6-132). Seven had been previously treated with other second line agents. Thirty-eight patients had an excellent or good response. In them, the erythrocyte sedimentation rate dropped in a mean of 38 mm/h. Only 2 patients had a rather poor response. Two patients discontinued the medication because of side effects: leukopenia in one and stomatitis in the other. Eleven patients presented with liver test abnormalities: 3 mild, 6 moderate and 2 severe. Seven patients had 11 liver biopsies. Except for one, none had evidence of cirrhosis or inflammation. Indeed, no changes were observed in the histopathology in those with repeated biopsies. The case reported as cirrhosis occurred very early in the course of MTX therapy. He continued taking MTX treatment without further deterioration of liver chemistry and/or histology. It is concluded that MTX is an effective and safe agent in PsA. Results also indicate that it is not necessary to perform liver biopsies on a routine basis.


Glucocorticoid receptor in patients with lupus nephritis: relationship between receptor levels in mononuclear leukocytes and effect of glucocorticoid therapy.

We investigated the clinical significance of glucocorticoid receptor determination in 20 patients with systemic lupus erythematosus (SLE) who afterwards developed nephrotic syndrome. Glucocorticoid receptor concentrations in mononuclear leukocytes (MNL) in these patients were comparable with those in both other patients with SLE and healthy persons. Improvement in urinary protein excretion and in disease activity, which was scored according to the SLE Disease Activity Index system of the University of Toronto, closely related to the glucocorticoid receptor concentrations in MNL isolated from the corresponding patients. In summary, glucocorticoid receptor determination in patients with lupus nephritis may be a predictive clue for assessing responsiveness to glucocorticoid therapy.


IgG autoantibody to the collagen-like region of Clq in hypocomplementemic urticarial vasculitis syndrome, systemic lupus erythematosus, and 6 other musculoskeletal or rheumatic diseases.

We previously found that the Clq precipitin in sera from patients with hypocomplementemic urticarial vasculitis syndrome is an IgG autoantibody to Clq. We report here a prevalence study of this autoantibody in 162 patients with musculoskeletal or rheumatic diseases including hypocomplementemic urticarial vasculitis syndrome, systemic lupus erythematosus (SLE), and rheumatoid arthritis uncomplicated by vasculitis. The autoantibody, which binds only to the collagen-like region of Clq, was found almost exclusively in hypocomplementemic urticarial vasculitis syndrome (100%) and SLE (35%) sera. Our results support the idea that among rheumatic diseases, anti-Clq autoantibody develops in disorders characterized by immune complex mediated injury, particularly of cutaneous and glomerular microvasculature.


A radiographic comparison of erosive osteoarthritis and idiopathic nodal osteoarthritis.

Clinical, radiographic and histologic features suggest that inflammation is central to the pathogenesis of erosive osteoarthritis (OA). Since mediators of inflammation may activate osteoclasts and stimulate release of metalloproteinases in joint cartilage, we hypothesized that patients with erosive OA may have more joint space narrowing and less proliferative bone response (osteophytes, sclerosis) than those with idiopathic nodal OA. Hand radiographs of 33 patients with erosive OA and 33 age and sex matched patients with nodal OA were evaluated for prevalence and severity of joint space narrowing, osteophytes, subchondral sclerosis, subchondral cysts, erosions and subchondral collapse. While the prevalence and severity of OA was greater at each joint in erosive OA than in nodal OA, significant differences (p less than 0.05) were confined largely to the interphalangeal joints. Among patients with erosive OA, radiographic features of OA were more severe in joints with erosive changes than in joints that did not show erosive change (p less than 0.01 in most cases). Notably, when joints with erosive change were excluded, only joint space narrowing was more severe in patients with erosive OA than in the corresponding joints of patients with nodal OA (p less than 0.001). Our analysis did not support the hypothesis that inflammatory mediators modify chondro or osteoneogenesis in erosive OA.


Effects of loading on the synthesis of proteoglycans in different layers of anatomically intact articular cartilage in vitro.

The anatomically intact articular cartilage (approximately equal to 2.5 cm2) of whole bovine sesamoid bones was cultured on its bone support. A load of 5 kg was applied intermittently at 0.3 Hz by a specially designed loading apparatus for 7 days. [35S]-sulfate was added during the last 17 h of the experiment. Loading induced a 40% increase in [35S]-sulfate incorporation into aggrecans and an almost 3-fold increase in the synthesis of small proteoglycans. The loading effects occurred mainly in the upper half of the articular cartilage. Samples left unloaded for 7 days and then loaded for 7 days likewise showed an increase in [35S]-sulfate incorporation compared with unloaded controls.


Bronchoalveolar lavage and transbronchial biopsy in spondyloarthropathies.

Fourteen patients (12 men, 2 women) with spondyloarthropathies underwent bronchoalveolar lavage (BAL), transbronchial biopsy (TBB) and other respiratory investigations. BAL revealed lymphocytosis and increased nonaltered neutrophil polynuclears in a smoker and an isolated lymphocytosis in one patient with restrictive syndrome and radiographic apical fibrosis. TBB showed interstitial fibrosis in the 2 patients and in 3 others who are all nonsmokers and among whom 2 had a restrictive syndrome. Subclinical alveolitis in spondyloarthropathies is absent. Interstitial fibrosis is not rare and its frequent association in a restrictive syndrome suggests a mechanical origin.


Followup in psoriatic arthritis: relationship to disease characteristics.

We investigated whether patient disease characteristics can explain differences in patients registered at the psoriatic arthritis clinic before 1987 who were seen regularly (139, "regular") to those who had not been seen for 2 years before 1989 (165, "inactive"). Despite a higher percentage of women in the regular group (p = 0.0058), logistic regression analysis identified no significant differences in disease severity or treatment at the first or last assessment before 1987. A followup study in 1989 revealed that of the 165 inactive patients, 20 had died and 64 had moved away. Of the 81 remaining patients, 18 were not interested, 18 were seeing other physicians, and 45 returned for followup. The latter 45 patients were compared to 85 of the regular patients who were seen during the same period. Both groups demonstrated similar disease attributes at first and last assessments before 1987. At their 1989 assessments, the same degree of disease progression was noted in both groups. Thus, loss to to followup appears to be random, and should not influence further studies of these patients.


Effect of flurbiprofen on cytokine production by human monocytes and U-937 and THP-1 cell lines.

Possible effects of nonsteroidal antiinflammatory drugs (NSAID) on inflammatory mediators other than arachidonic acid metabolites which might contribute to the antiinflammatory effects of these drugs have not been fully explored. We investigated the effects of an NSAID, flurbiprofen, on production of the cytokines tumor necrosis factor alpha (TNF alpha), interleukin 1 beta (IL-1 beta) and interleukin 6 (IL-6) by human peripheral blood monocytes and by the human cell lines U-937 and THP-1. Cytokine production was induced by 1 microgram/ml bacterial lipopolysaccharide (LPS) in both monocytes and cell lines, and cytokine levels in supernatants were measured by enzyme immunoassay. In monocytes, IL-6 was the major product while in both cell lines, TNF alpha was the major product. Flurbiprofen caused moderate inhibition of IL-1 beta and TNF alpha production by stimulated monocytes, but did not affect IL-6 production. In contrast, flurbiprofen completely abolished IL-6 production by both cell lines and substantially inhibited IL-1 beta and TNF alpha production. These observations raise the possibility that inhibition of cytokine production by flurbiprofen may contribute to the antiinflammatory properties of this drug.


Tetracyclines suppress matrix metalloproteinase activity in adjuvant arthritis and in combination with flurbiprofen, ameliorate bone damage.

Tetracyclines are potent inhibitors of 2 major matrix metalloproteinases which have been implicated in connective tissue degradation: collagenase and Type IV collagenase/gelatinase. We directly identified these enzyme activities in extracts of inflamed paw tissue from rats with adjuvant arthritis. Oral tetracycline therapy suppressed metalloproteinase activity in arthritic tissue, but even very high doses failed to exhibit substantial antiinflammatory efficacy (reduced joint swelling and paw diameter). Flurbiprofen, a conventional nonsteroidal antiinflammatory drug, reduced inflammatory indices as expected. The combination of the 2 agents administered orally completely inhibited collagenase activity, significantly inhibited gelatinase activity and produced substantial normalization of radiographic joint damage, far greater than either drug alone. Tetracycline inhibition curves in vitro suggest that the collagenase in this tissue is not of fibroblast origin. Tetracycline derivatives might be useful adjuncts to prevention of tissue damage in chronic inflammatory arthritides.


The fibromyalgia and myofascial pain syndromes: a preliminary study of tender points and trigger points in persons with fibromyalgia, myofascial pain syndrome and no disease.

Four experts on myofascial pain syndrome (MFP) performed trigger point examinations and 4 experts on fibromyalgia performed tender point examinations on 3 groups of subjects (7 patients with fibromyalgia, 8 with MFP, and 8 healthy persons) while blinded as to diagnosis. Local tenderness was common in both disease groups (65-82%), but was elicited in a greater proportion of MFP experts' examinations (82%). Active trigger points were found in about 18% of examinations of patients with fibromyalgia and MFP, but latent trigger points were rare in all groups. A more liberal definition of trigger point, however, resulted in a 38 and 23% positive rate among patients with fibromyalgia and MFP, respectively. Taut muscle bands and muscle twitches were common (50 and 30%, respectively) and noted equally in all 3 diagnostic groups. Problems with reliability were identified for taut bands, muscle twitch, and active trigger points. Our data are exploratory and tentative, but suggest that attention to definition and reliability are required to advance our knowledge of these common syndromes.


The economic impacts of juvenile rheumatoid arthritis.

Our study documents the direct costs, family costs and community (extra school) costs. One hundred and twenty families with children who had juvenile rheumatoid arthritis (JRA) diagnosed by established criteria and who lived in New England were asked to participate. All data except inpatient charged were collected via questionnaire. The questionnaire return rate was 59% (N = 70). The mean annualized direct cost/child was 7,905 (inpatient, $1,717; outpatient, $5,700; and nonmedical, $488). Family costs averaged $1,524/year (out of pocket medical and nonmedical, $1,196; lost salary, $328), which represented 5% of mean family income. The mean extra school cost was $1,449/9 months. The economic impacts of JRA appear to be substantial.


Intracranial findings in progressive facial hemiatrophy.

There have been infrequent reports of cerebral lesions associated with progressive facial hemiatrophy. Six children with progressive facial hemiatrophy were evaluated. Four were referred for evaluation of neurological deficits: 2 with seizures, one with left hemiparesis and one with learning problems. The remaining 2 patients had only facial hemiatrophy. Cranial computed tomography (CT) in 5 patients revealed the bony and soft tissue defects, but cerebral calcifications were seen in only 3 patients. Cranial magnetic resonance imaging (MRI) demonstrated areas of increased signal in the ipsilateral white matter on T2 weighted images in all 5 patients with upper facial atrophy. Ipsilateral cerebral lesions with progressive facial hemiatrophy may be more common than once believed. MRI sometimes reveals abnormalities of the white matter even in patients without neurologic symptoms, and may be more sensitive than CT in the diagnostic evaluation of patients with progressive facial hemiatrophy.


Antibodies to lipid A in pauciarticular juvenile arthritis: clinical studies.

IgG antibodies to monophosphoryl lipid A (MPL) are more concentrated in synovial fluids than in the blood of children with pauciarticular juvenile arthritis. Correlations between IgG anti-MPL levels and numbers of inflamed joints were highly significant in 2 of 10 patients followed for 8 years and suggestive in 2 patients followed for shorter periods. A fifth patient had a correlation between IgM anti-MPL and joint count.


Exocrine pancreatic function in children with systemic lupus erythematosus.

We examined the incidence and spectrum of pancreatic disease in pediatric systemic lupus erythematosus (SLE). We measured serum immunoreactive cationic trypsinogen (IRT) in 185 samples obtained from 35 patients with SLE. Fifteen patients (43%) had elevated IRT levels on at least one occasion. Serum samples were obtained in 20 of 35 patients before start of treatment. Seven of these 20 patients (35%) had elevated IRT levels at diagnosis, which slowly returned to normal as their disease was controlled with treatment. A further 3 of these 20 patients in whom we had sera at diagnosis had elevated levels at some course during their illness. Of the remaining 15 patients in whom sera was not available at diagnosis, 5 patients had increased IRT levels on at least one occasion. We show that elevated IRT levels are common in pediatric SLE, but there was no apparent association with drugs such as prednisone and azathioprine. However, high levels of IRT at the time of diagnosis may be related to an underlying disease component such as vasculitis.


Childhood linear scleroderma: a possible role of thermography for evaluation.

Linear scleroderma is a rare, at times debilitating, disease with an unpredictable course. Currently, there is no satisfactory objective method for assessment of disease activity upon which to base therapeutic decisions. We evaluated 11 children with 18 linear scleroderma lesions (mean age 11.7 years, mean duration of disease 5.1 years) for disease severity and the presence of immunologic abnormalities, and attempted to correlate these results with thermography. Positive thermography was defined as warmer than surrounding skin or opposite limb by 0.5 degrees C. Six patients were thermography positive. Mean age, sex, disease duration and the presence of hypergammaglobulinemia and autoantibodies were similar in thermography positive and thermography negative patients. Six of 18 linear scleroderma lesions were thermography positive. All 3 new or expanding lesions were thermography positive. All 3 lesions that were resolving clinically were thermography negative. Three of 12 lesions that were clinically unchanged over a 6-month period were also thermography positive. In summary, thermography is a noninvasive test that appears to demonstrate active lesions in linear scleroderma. It is not influenced by previous soft tissue damage induced by linear scleroderma and may enable better monitoring of the effectiveness of proposed therapies.


Upper airway obstruction as the sole manifestation of rheumatoid arthritis.

A 51-year-old woman with a history of rheumatoid arthritis (RA) suddenly developed laryngeal stridor. Rheumatoid factor was positive and the erythrocyte sedimentation rate was elevated, but she had no evidence of acute peripheral arthritis at this time. A flexible laryngoscopy revealed cricoarytenoid arthritis which was the sole clinical manifestation of active RA. Involvement of the cricoarytenoid joints must be considered in patients with RA with laryngeal stridor even in the absence of active peripheral arthritis.


Drug induced systemic lupus erythematosus due to ophthalmic timolol.

We report a case of systemic lupus erythematosus (SLE) apparently induced by topical use of ophthalmic timolol maleate, a beta adrenergic blocking agent. The patient developed fever, malaise, pleurisy and recurrent sterile pleural effusions while taking no medication other than timolol. Antinuclear antibodies in a homogenous pattern, and markedly elevated histone antibodies (IgG anti-(H2A-H2B)-DNA) were present while antibodies to native DNA were absent. After discontinuation of the timolol, his symptoms improved promptly and the pleural effusions resolved. To our knowledge, this is the first report of timolol induced SLE.


Jaccoud's arthropathy of the shoulders in systemic lupus erythematosus.

A man with longstanding systemic lupus erythematosus who developed anterior dislocation of both shoulder joints is described. He also had typical Jaccoud's deformities of his hands and feet.


Systemic lupus erythematosus and pheochromocytoma.

A 40-year-old Chinese woman with systemic lupus erythematosus (SLE) developed labile blood pressure 3 years after her onset of high blood pressure. Pheochromocytoma was subsequently found and removed. Postoperatively her hypertension improved without change in her SLE activity. Our case demonstrates pheochromocytoma can be a cause of hypertension in SLE.


Prosthetic knee infection due to Achromobacter xylosoxidans.

Achromobacter xylosoxidans is an aerobic gram negative organism that has been infrequently implicated in clinical infections in a variety of anatomical sites. We describe a case of a prosthetic knee infection due to Achromobacter xylosoxidans in a patient with rheumatoid arthritis receiving high dose prednisone.


Heterotopic ossification presenting as acute arthritis.

We describe a patient with acute myeloblastic leukemia in remission who developed painful, swollen knees 6 weeks after being ventilated for acute respiratory distress syndrome. Synovial fluid analysis was nondiagnostic and initial radiographs of his knees were normal. Repeat radiographs 5 weeks later showed periarticular calcification and the diagnosis of heterotopic ossification was made. Heterotopic ossification should be part of the differential diagnosis of acute arthritis in critically ill patients.


Renal involvement in primary antiphospholipid syndrome.

Our objective was to define the renal involvement in primary antiphospholipid syndrome (APS). We studied 20 patients with primary APS. Fourteen were women, mean age 34.4 years. None met ARA criteria for systemic lupus erythematosus. All patients underwent complete renal function studies. The presence of hypertension was also investigated. Renal disease was found in 5 patients, and was characterized by proteinuria, hypertension and renal failure. Kidney biopsy was performed in these 5 patients, showing thromboses of the microvasculature, mesangiolysis, mesangial interposition, electron lucent subendothelial material and ischemic obsolescence of glomeruli. Arterioles showed luminal narrowing due to medial hypertrophy, mucoid thickening of the intima, thrombosis and fibrosis. We found renal disease in 25% of our patients with primary APS. Biopsy findings were consistent with a thrombotic microangiopathy involving both arterioles and glomerular capillaries.


Upper airway disease in systemic lupus erythematosus: a report of 4 cases and a review of the literature.

We report the occurrence of life threatening upper airway complications in 4 of 158 patients with systemic lupus erythematosus (SLE) admitted to our hospital over a 6-year period and review published reports on this complication. The patients included in our study were identified through a computer search of our hospital medical records. Patients' charts were reviewed and the diagnosis confirmed by currently accepted criteria. The clinical features of SLE in all the published cases were assessed and compared to those found in our patients. We found that although upper airway involvement is uncommon in SLE, occurring with a relative frequency of 4/158 patient admissions, endotracheal intubation in patients with active SLE may result in the development of life threatening upper airway complications. We suggest that patients with active SLE who require intubation should be observed for the development of upper airway symptoms.


Patients with systemic lupus erythematosus at risk for Pneumocystis carinii pneumonia.

We describe 6 cases of patients with systemic lupus erythematosus (SLE) who developed Pneumocystis carinii pneumonia. All were treated with high dose corticosteroids, and all developed the infection within 4 months of beginning new or revised cytotoxic therapy. All patients tested (5 of 6) were negative for human immunodeficiency virus (HIV). Those patients who developed Pneumocystis carinii pneumonia had more severe lymphocytopenia (median 595 vs 833/mm3) and received higher doses of corticosteroids (median prednisone dose = 43 vs 20 mg/day) than other patients with active SLE. A threshold lymphocyte count of 350/mm3 identified 4 of 6 cases but only 1 of 20 controls. Patients with SLE treated with high dose corticosteroids and cytotoxic drugs and with severe lymphocytopenia may be at increased risk for this opportunistic infection.


Macrophage infiltration in the skin of patients with systemic sclerosis.

We stained skin specimens from 31 patients with systemic sclerosis with monoclonal antibodies to macrophages and T lymphocytes to characterize the cellular infiltrates. Not only T lymphocytes but macrophages were also identified, and macrophages were observed between collagen bundles as well as around skin adnexa and vessels. In addition, the ratio of macrophages to T lymphocytes was high, particularly in severely affected skin. There was no close relationship with disease duration. Our results suggest that cutaneous macrophages always play a role in scleroderma.


Mechanisms of pathogenesis in scleroderma. I. Overproduction of interleukin 6 by fibroblasts cultured from affected skin sites of patients with scleroderma.

Fibroblasts were cultured from affected skin sites of patients with progressive systemic sclerosis (PSS), from unaffected skin sites of the same patients, and from a healthy donor. The concentration of interleukin 6 (IL-6) in culture medium conditioned by the growth of early passage cells was determined by radioimmunoassay and by quantitative bioassay. Results demonstrated that fibroblasts from affected PSS skin produce from 6 to 30-fold higher levels of biologically active IL-6 compared to unaffected and control cells. In contrast, serum IL-6 concentrations in 6 of 8 patients examined were not significantly different from healthy donors. Serum IL-6 levels were elevated 2 to 3-fold in 2 of 8 patients examined. Thus, the overproduction of IL-6 by affected scleroderma fibroblasts does not necessarily correlate with a systemic increase in IL-6, but may increase its concentration locally. In view of its biological activities, including stimulation of antibody production and T cell activation, the overproduction of IL-6 by PSS fibroblasts in the lesions may play a significant role in the pathogenesis of PSS and may profoundly influence the course of the disease.


Mechanisms of pathogenesis in scleroderma. II. Effects of serum and conditioned culture medium on fibroblast function in scleroderma.

Scleroderma (progressive systemic sclerosis; PSS) is a connective tissue disorder in which excessive collagen is deposited in the skin and internal organs. Mediators of abnormal fibroblast function in PSS have not yet been identified. Our objective was to examine the possibility that factors present in serum from patients with PSS, or in culture medium conditioned by PSS fibroblast growth, serve to regulate fibroblast function. Fibroblasts from affected and unaffected skin sites of patients with PSS and from normal adult skin were cultured in the presence of various human sera or conditioned media. Results indicate that (1) the proliferative influence of serum of patients with PSS is not different from that of normal donors, (2) increased proliferation and procollagen gene expression are not linked in affected or unaffected dermal fibroblasts, and (3) affected PSS fibroblasts produce a stimulatory factor(s) for procollagen gene expression to which they are differentially sensitive.


Management of suspected giant cell arteritis: a decision analysis.

We used decision analysis to compare 4 strategies for managing suspected giant cell arteritis (GCA): treat no patients, treat all patients, perform temporal artery biopsy and only treat positive cases, or biopsy and treat all cases irrespective of the result. The "treat no patients" strategy is preferred only if the likelihood of disease is less than 2%. The "biopsy and treat positive cases" strategy predominates for intermediate likelihood of disease (50%) and is insensitive to altering the probability of any key variables. The "biopsy but treat irrespective of the result" strategy becomes preferred at likelihood of disease of 81%. However, this result was sensitive to alteration of the key variables. Empirical therapy without biopsy would only be preferred if the likelihood of disease exceeded 90% and the disutility one attaches to treating under uncertainty is low at high probability of disease. Management of suspected GCA should be guided by the results of temporal artery biopsy in the majority of cases. At high likelihood of disease, temporal artery biopsy may be of value independent of its usefulness in determining management strategy. Further research should be directed towards improving the accuracy of diagnosis, defining the risk of iatrogenic complications and estimating utilities for the outcomes.


Character of precolumbian North American spondyloarthropathy.

Shared characteristics and concurrent occurrence of rheumatoid arthritis (RA) and spondyloarthropathy in contemporary populations have compromised development of clear diagnostic criteria for distinguishing them. Although modern populations contain individuals with both RA and spondyloarthropathy, ancient populations often manifest only one. The presence of spondyloarthropathy as the sole erosive disease in selected ancient populations allows further clarification of its nature. The tendency towards pauciarticular, asymmetrical involvement, axial involvement and peripheral joint fusion in these populations clarifies diagnosis and distinguishes this phenomenon from RA. The significance of peripheral joint fusion appears to be unequivocally established on the basis of these findings.


Pathogenetic role of Chlamydia, Yersinia and Borrelia in undifferentiated oligoarthritis.

We studied the cellular and humoral immune response to Chlamydia trachomatis, Yersinia enterocolitica and Borrelia burgdorferi in paired samples of peripheral blood and synovial fluid (SF) in undifferentiated oligoarthritis, reactive arthritis (ReA) and rheumatoid arthritis. Antigen specific lymphocyte proliferation was found in SF of 43% of patients with ReA and 34% of patients with undifferentiated oligoarthritis. C. trachomatis was the most frequent single agent. HLA-B27 was positive in 83% of patients with ReA and in 62% of patients with undifferentiated oligoarthritis with antigen specific lymphocyte proliferation. Antigen specific lymphocyte proliferation correlated poorly with the specific antibody response. Only chlamydial antigen was detected in SF cells using monoclonal antibodies. We conclude that some patients with undifferentiated oligoarthritis may have a forme fruste of ReA. This finding is important in view of recent evidence supporting the efficacy of antibiotic therapy in ReA.


HLA-DP restricted Chlamydia trachomatis specific synovial fluid T cell clones in Chlamydia induced Reiter's disease.

Synovial fluid (SF) mononuclear cells from a patient with Chlamydia trachomatis induced acute Reiter's disease were directly by limiting dilution in a representative protocol using phytohemagglutinin in the cloning medium. Out of 76 alpha beta-TCR+ CD4+ T lymphocyte clones, 7 were shown to specifically recognize C. trachomatis in a proliferation assay. The antigen recognition of these clones was HLA-DP restricted. Unexpectedly, 2 HLA-DR restricted clones showed a proliferative response to Yersinia enterocolitica O3, though the patient had no history of yersinia infection. The high frequency of SF derived T cells with specificity for species-specific chlamydial antigens and the limited diversity of HLA class II restriction of these clones may indicate an oligoclonal synovial T cell response to persistent intraarticular chlamydia.


Prevalence of cartilage shards in synovium and their association with synovitis in patients with early and endstage osteoarthritis.

It has been suggested that incorporation of shards of fibrillated cartilage into the synovium is a cause of synovitis in osteoarthritis (OA). We examined the prevalence with which fragments of cartilage are seen in synovium, and their association with synovitis, in patients with endstage OA and early OA of the knee. Samples of synovium were obtained from 12 patients with endstage OA who were undergoing knee joint replacement and 30 with only mild/moderate radiographic changes of OA who exhibited articular cartilage changes of OA at arthroscopy. The presence of cartilage shards was sought in synovium from the medial and lateral tibiofemoral compartments and the suprapatellar pouch of each patient. Comparable volumes of the synovial lining from patients with endstage and early OA were examined, and tissue mononuclear cell infiltration was graded as an indicator of synovitis. Cartilage shards were seen in synovium from 7 of 12 patients with endstage OA, all of whom had synovitis. No topographic relationship was found between shards and mononuclear cell infiltration. In contrast, cartilage fragments were not seen in synovium from any of the 30 patients with early OA, although 9 of them had full thickness cartilage ulcers and 17 had evidence of synovitis.


Predicting outcome from intensive care for patients with rheumatologic diseases.

To evaluate 2 prognostic scoring systems in patients with an underlying rheumatologic diagnosis admitted to an intensive care unit (ICU). A retrospective case series review, carried out at a medical ICU in a military referral hospital. All adult ICU admissions with a known rheumatologic diagnosis were evaluated during 28 consecutive months. There were 48 ICU admissions available for review in 36 patients (1.33 ICU admissions/patient) during the study period. All patients were assigned an APACHE II and TISS score based on the first 24 h of ICU admission. Eleven ICU admissions resulted in patient death (22.9%) and the remaining 37 admissions (77.1%) in patient survival and hospital discharge. Overall patient mortality was 30.6% for the study population. The APACHE II and TISS scores were each significantly different for survivor and nonsurvivor subgroups (APACHE II p less than 0.0001; TISS p less than 0.0001). In this group of patients evaluated at a single institution both the APACHE II and TISS scoring systems allowed subgroup separation between survivors and nonsurvivors of ICU admission. However, these scoring methods demonstrated limitations in terms of outcome prediction when applied to the individual patient.


Secondary avascular necrosis in coxarthrosis: a morphologic study.

Secondary subarticular avascular necrosis was observed grossly in 11.7% of femoral heads removed surgically because of osteoarthritis (OA). In most cases the necrosis was superficial, however in 15% of the 82 cases of secondary necrosis the necrosis was deep and wedge shaped similar in appearance to the lesions seen in primary subarticular avascular necrosis. Most of the lesions were seen in the early stage of OA and it is suggested that the lesion may interfere with the subsequent reparative phenomenon.


HLA antigens in primary fibromyalgia syndrome.

HLA antigens, both class I (A,B,C) and II (DR), were determined in 60 patients with primary fibromyalgia syndrome and compared to 159 healthy controls. No significant association between primary fibromyalgia syndrome and alleles of the HLA system was detected.


The arthropathy of hereditary arthroophthalmopathy (Stickler syndrome).

Hereditary arthroophthalmopathy (Stickler syndrome) is an autosomal dominant syndrome characterized by musculoskeletal, ophthalmic and dysmorphic facial features. A family is described illustrating diverse expressions of Stickler syndrome, including abnormalities not directly attributable to mutation of the type II procollagen gene. A review of the literature demonstrates a range of articular problems, several of which are not specific to Stickler syndrome, and might be encountered in either adult or pediatric rheumatology practice. Stickler syndrome may be underrecognized by rheumatologists, particularly if the significance of nonarticular clinical features or a positive family history are not appreciated.


A nutritional screening test for use in children and adolescents with juvenile rheumatoid arthritis.

Protein-energy malnutrition (PEM) has been demonstrated in about 35% of patients with juvenile rheumatoid arthritis (JRA), but fewer than 8% of children with rheumatic diseases were reported in a national survey to have been seen by a pediatric dietitian. We demonstrate the development of a nutritional screening test for PEM in patients with JRA for use by all health care professionals. Nutritional assessment of 74 patients with JRA was conducted using a standardized 11 variable profile comprised of upper body anthropometric and biochemical measurements. The sensitivity, specificity, predictive values and index of validity were calculated for individual and selected clusters of nutritional variables to predict the need for referral for PEM compared to the independent review by 2 pediatric dietitians to refer or not refer to a dietitian for further evaluation or care. Arm circumference less than or equal to 10th percentile for age and sex matched norms was selected as the screening test for PEM in patients with JRA due to a combination of excellent measurement characteristics (sensitivity 0.80, specificity 0.86, positive predictive value 0.90, negative predictive value 0.73, index of validity 0.88) and ease of measurement.


SEA syndrome revisited: a longterm followup of children with a syndrome of seronegative enthesopathy and arthropathy.

Thirty-six of the 39 children originally described with the syndrome of seronegative enthesopathy and arthropathy, followed for a mean of 11 years after symptom onset, were found to have had a widely varied clinical course. Twelve of the 23 patients (52%) who originally did not have a seronegative spondyloarthropathy developed definite (6) or possible (6) seronegative spondyloarthropathies. The development of a seronegative spondyloarthropathy was associated with HLA-B27 (p = 0.0004) and the presence of arthritis (rather than arthralgia only) at the time of the original report (p = 0.05). For patients with arthritis, the development of a seronegative spondyloarthropathy was associated with arthritis onset after 5 years of age (p = 0.01).


Primary antiphospholipid syndrome with multiorgan arterial and venous thromboses.

A 57-year-old man with no evidence of infection, vasculitis or connective tissue disease died with multiple organ thromboses after an acute illness. He was found to have lupus anticoagulant, IgG anticardiolipin antibody, false positive rapid plasma reagin, prolonged partial thromboplastin time, and thrombocytopenia. Venous and arterial thrombi leading to necrosis were found in his scrotum, testicles, upper and lower extremities, adrenals, kidneys, lungs, and brain. No other explanation could be found for his fatal illness, thus suggesting the primary antiphospholipid syndrome (APS). This is a documented case of primary APS associated with multiorgan arterial and venous thromboses of large and small vessels, presenting as a fulminant and fatal acute illness.


Intracardiac mural thrombus mimicking atrial myxoma in the antiphospholipid syndrome.

A wide spectrum of cardiac involvement including valvular lesions, myocardial infarction and myocardial dysfunction has been reported in patients with antiphospholipid antibodies (aPL), suggesting that cardiac manifestations may be part of the antiphospholipid syndrome (APS). We describe 3 patients (2 with primary APS and one with APS and SLE) who were found to have right atrial masses by echocardiography (transthoracic and/or transesophageal) and/or angiography, which were felt preoperatively to be atrial myxomata. Pathological examination of resected material showed only organized thrombus with calcification. We describe outcome 12 months to 7 years after resection of thrombus and document possible recurrence in one patient after 7 years. Intracardiac mural thrombus may be an additional cardiac manifestation of the APS, and presents considerable diagnostic confusion in its differentiation from atrial myxomata.


Communicating hydrocephalus in systemic lupus erythematosus with antiphospholipid antibody syndrome.

We report a case of communicating hydrocephalus in a 24-year-old woman with previously undiagnosed systemic lupus erythematosus (SLE) presenting with malignant hypertension, nephritis, serositis, and a seizure disorder of 16 months' duration. The patient demonstrated features of the antiphospholipid antibody syndrome (APS). In proposing cerebral venous thrombosis as a possible, yet unproven, pathophysiologic mechanism for the hydrocephalus in this case we have reviewed and summarized literature relating to SLE, APS, hydrocephalus and pseudotumor cerebri. In cases of unexplained pseudotumor cerebri or hydrocephalus, a search for SLE and APS should be considered.


Systemic lupus erythematosus presenting with myelofibrosis.

The 20-year-old girl we describe presented with myelofibrosis and systemic lupus erythematosus (SLE), which initially responded to treatment with corticosteroids but during a relapse 9 months later a bone marrow biopsy revealed no improvement in her myelofibrosis. As the cytotoxic treatments used to treat severe SLE may be associated with bone marrow suppression, it is important to consider the possibility of myelofibrosis complicating SLE, which, even when it is a presenting feature, may not readily respond to corticosteroids. When this girl was subsequently treated with high dose steroid and azathioprine her myelofibrosis went into remission.


Streptococcus pyomyositis occurring in a patient with dermatomyositis in a country with temperate climate.

We describe a man in whom pyomyositis developed in a temperate climate. Three facts make this case unique. First the pyomyositis developed in someone with underlying dermatomyositis, this being the second reported case to our knowledge. Second, the organism involved was a Streptococcus and not a Staphylococcus as in most cases described, and the course of the disease was acute and not subacute as is usually reported. Finally, contrary to most described cases, surgical drainage was not necessary, probably because of the early diagnosis. Pyomyositis should be considered a possible cause of localized pain in patients with underlying inflammatory muscle disease.


Concurrent temporal arteritis and Churg-Strauss syndrome.

We describe the case of a 41-year-old man who presented with clinical and histopathologic evidence of temporal artery lesions associated with the Churg-Strauss syndrome. Pathological examination of the temporal artery showed panarteritis without giant cell formation or fibrinoid necrosis. We review the world literature concerning the vasculitides with features that overlap giant cell arteritis (GCA) and polyarteritis nodosa (PAN) and classify into 2 sub-groups PAN with unusual temporal artery localization and GCA with variably disseminated arterial injuries. These cases emphasize the fact that not all arteritis involving the temporal arteries is GCA. Only 3 cases with temporal artery involvement and concurrent Churg-Strauss syndrome have been published.


General practitioners' use of non-steroidal anti-inflammatory drugs in Tayside and Fife regions.

The objectives of this study were to assess the prescribing of non-steroidal anti-inflammatory drugs (NSAIDs) by general practitioners and to determine their attitudes to problems caused by this class of drugs. The study consisted of two parts. The first was a questionnaire survey among general practitioners in Fife and Tayside, and the second was an analysis of NSAID prescribing over 12 months among the doctors in the Carnoustie Health Centre, using duplicate prescriptions. In the questionnaire survey 61% of the general practitioners responded. The three most preferred drugs were buprofen (56%), naproxen (20%) and mefenamic acid (7%); choice of drug was determined by efficacy and personal experience. Gastrointestinal side effects were most frequently encountered, although there was little consensus amongst respondents as to their management. The duplicate prescription study showed that 14% of patients (1607 individuals) received at least one NSAID prescription in the year of study. Ibuprofen (31%), naproxen (20%) and piroxicam (15%) were most frequently prescribed and up to 16% of the patients were co-prescribed a gastroprotective agent; ranitidine (75%) was the most commonly prescribed. Despite the introduction of newer NSAIDs, ibuprofen and naproxen are still the most commonly prescribed drugs. Furthermore, although gastrointestinal side effects are commonly encountered, there is some uncertainty about their management.


Prevalence of mitral valve prolapse in keratoconus patients.

Fifty patients with advanced degrees of keratoconus, requiring corneal transplantation, were screened for mitral valve prolapse by two dimensional echocardiography. The overall prevalence of 58% was found to be statistically higher than the prevalence of 7% found in a group of age and sex-matched controls. It was also found to be higher than the previously reported prevalence of 38% in a group of keratoconus patients with similar age and sex match to our series. The findings of our study in conjunction with the histopathological and biochemical similarities between the two conditions strongly suggest that they may be different manifestations of similar defects in collagen metabolism.


Cerebellar ataxia in the elderly.

In a retrospective study of 624 elderly patients referred with falls and gait disorders, 45 patients were found to have ataxia. Cerebrovascular diseases were the most common underlying cause of ataxia (15 patients, 37%). Nine patients had hereditary/degenerative cerebellar ataxia. History suggesting alcohol as an underlying cause was established in two patients with cerebellar ataxia. Three patients had normal pressure hydrocephalus and their condition improved remarkably after surgery. No definite cause was found in five patients. Cranial computed tomography (CT) showed cerebral atrophy in 16 patients and in three patients there was evidence of atrophy of the cerebellar vermis. Four patients had femoral neck fractures and three patients had other fractures. In a 5-year follow-up five patients died with bronchopneumonia (11% mortality) and patients with dementia showed rapid deterioration. All patients were referred to the day hospital for rehabilitation. The best treatment outcome was achieved in patients who had a single cerebrovascular accident with no cognitive impairment and in those whose ataxia was secondary to medication. Fourteen patients (44%) moved to residential care while 27 (66%) continued to live in their homes with community support. We concluded that there is no evidence of increased mortality in the elderly patients with cerebellar ataxia. CT scan is mainly helpful in diagnosing specific diseases such as tumours or hydrocephalus. A significant proportion of elderly patients with ataxia may have reversible or treatable conditions and can pursue an independent life.


Cardiopulmonary resuscitation training revisited.

The cardiopulmonary resuscitation (CPR) skills of 50 junior hospital doctors (22 house officers and 28 senior house officers) were assessed. Theoretical knowledge was measured by a multiple choice questionnaire and practical ability with the Laerdal Skillmeter Resusci Anne. Only 40% of the study group passed both tests. Those doctors who had previously received regular CPR training performed better in the practical test (P<0.05) than those who had not. Theoretical knowledge was unrelated to previous CPR training. It is recommended that junior hospital doctors should undergo regular CPR training every 6 months, in order to maintain their practical CPR skills.


Loco-regional recurrence and survival after wide local excision, radiotherapy and axillary clearance for early breast cancer.

After breast conservation for early breast cancer which comprised wide local excision, axillary clearance and radiotherapy to the breast, 145 women have been followed prospectively for a median of 42 months. Local recurrence occurred in 11 (7%) and axillary recurrence in three (2%). Distant recurrence has occurred in 32 and accounted for 80% of all first recurrences. Local treatment failure has occurred in three women and would not have been prevented by mastectomy. Loco-regional recurrence in the absence of preceding or synchronous distant disease was unusual and did not pose a significant clinical problem.


Dental enamel growth, perikymata and hypoplasia in ancient tooth crowns.

This paper describes the hypoplastic defects commonly seen on the surface of ancient human tooth crowns, excavated from archaeological sites, and presents a new method for estimating the ages at which these defects were initiated during life. The method is based upon examination of microscopic incremental structures on the enamel surface and it is possible also to apply it to reconstruction of the sequence and timing of dental crown development. The method of examination is non-destructive and allows full use to be made of the large numbers of complete, unworn dentitions which are found amongst archaeological remains.


Functional anatomy of the penguin flipper.

This study investigates the functional anatomy of the flipper of the jackass penguin (Speniscus demersus). The flippers of birds (n = 15) were dissected and described. Several adaptations from the typical avian wing were noted and reasons were proposed for these. The conclusion is that the osteological and myological adaptations of the flipper are designed to enable the penguin to progress very effectively through water, while the vascular adaptations provide a highly efficient mechanism for thermoregulation.


A qualitative study of the attitudes of members of a developing community towards their dogs and veterinary services.

A university-based hospital serving mainly a developing community readily appreciated that its clientele was from a very different sociological and cultural background than that of the professional staff and that developed and developing communities and their pets exhibit different patterns in their health status, with most diseases being predominantly related to socio-economic predicament. The need for a community-orientated service was recognised. From 1987 to 1988, the small animal outpatient clinic was used for an informal, unstructured sociological observation and interview-based study of the clientele and their pets. During this period, an informal educational programme was implemented to teach basic pet health care, and the efficacy of this programme was empirically gauged by comparing the knowledge and attitudes of first-time clients with those of regular clients. Regular clients appeared to become increasingly concerned about the health and management of their pets and the condition of their animals appeared to improve, with a corresponding decline in the degree of parasitism. The majority of clients however, still regarded the main reason for keeping dogs as the provision of some form of security. It also appeared as if improved attitudes towards household pets paralleled improvements in socio-economic status.


Tick control on cattle with flumethrin pour-on through a Duncan Applicator.

Cattle were treated for tick infestation using 1% flumethrin pour-on applied through a self-medicating device known as the Duncan Applicator, in 4 situations in Zimbabwe. Moderate to heavy tick infestations were successfully controlled on cattle.


The diagnosis and treatment of bovine genital ureaplasmosis: a case study.

An outbreak of granular vulvitis and ulcerative posthitis in 24-month-old virgin Bonsmara heifers and bulls is reported. Ureaplasma was isolated locally from all the clinically infected cattle. There was marked clinical improvement within 3 d of the commencement of a 5 d course of tylosin administered intramuscularly at 10 mg kg-1. Ureaplasma could not be cultured from the external genitalia of either heifers or bulls following clinical recovery. Details of the Ureaplasma culture media are given.


Anaplastic tracheal squamous cell carcinoma in a cat.

A cat with primary intratracheal squamous cell carcinoma presented with severe dyspnoea and a dry cough. Radiographic changes included pulmonary hyperinflation and tracheal thickening at the thoracic inlet. On post mortem examination, proliferative, white, granular nodules in the trachea were confirmed to be an anaplastic squamous cell carcinoma.


Clinical and biochemistry findings, and parathyroid hormone concentrations in three horses with secondary hyperparathyroidism.

Three cases of horses with nutritional secondary hyperparathyroidism (NSH) are described. The horses showed typical thickening of the maxillae and mandibular bones with or without lameness. Laboratory findings included elevated concentrations of parathyroid hormone (carboxy-terminal and mid-molecule fractions), alkaline phosphatase and an increase in the fractional excretion rate of serum inorganic phosphorus.


Synthesis and antimalarial properties of 1-imino derivatives of 7-chloro-3-substituted-3,4-dihydro-1,9(2H,10H)-acridinediones and related structures.

To improve upon the activity and properties of the 3-aryl-7-chloro-3,4- dihydro-1,9(2H,10H)-acridinediones, a variety of 1-[(alkylamino)alkylene]imino derivatives (3) were prepared and shown to be highly active antimalarial agents in both rodents and primates. Among structural modifications prepared, including N10-alkyl and C2-substituted analogs, removal of the C9 oxygen, and introduction of an imino side chain at C9, the imines of the N10-H acridinediones were the most active compounds obtained. The [3-(N,N- dimethylamino)propyl]imino derivative of 7-chloro-3-(2,4-dichlorophenyl)-3,4-dihydro-1,9(2H,10H)- acridinedione (9aa) proved to be highly active in advanced studies in primates.


Acyclic analogues of lipid A: synthesis and biological activities.

The synthesis of a series of novel acyclic analogues of lipid A, the lipophilic terminal of lipopolysaccharide (LPS), is reported. In these compounds, the reducing glucose unit of lipid A has been replaced by an acyclic analogue unit (abbreviated as AAU) consisting of a spacer (of varying length), an (R)-3-hydroxytetradecanamido moiety (of varying configuration at the carbon of attachment), and a CO2H group. The AAU has been attached to the anomeric carbon of the nonreducing glucose unit of lipid A, either through glycosidic linkage or through an acyl linkage. Further, amide isosteres of these acyclic analogues have been prepared using suitably protected 2,3-diamino-2,3-dideoxyglucose instead of 2-amino-2-deoxyglucose. All the compounds were well characterized and were tested for their ability to induce TNF-alpha in mouse bone marrow-derived macrophages, to enhance nonspecific resistance to infection in mice and to induce endotoxic shock in mice. The results showed a dramatic dependence, for the first time, on the length of the spacer and on the configuration of the carbon bearing the amido group in the AAU part of the analogues.


Synthesis and antibacterial activity of 1-(substituted pyrrolyl)-7-(substituted amino)-6-fluoro-1,4-dihydro-4-oxo-3- quinolinecarboxylic acids.

Seventeen quinolone compounds characterized by having a fluorine atom at the 6-position, a substituted amino at the 7-position, and a substituted pyrrolyl at the 1-position were synthesized for the first time. The in vitro antibacterial activities of these compounds against Escherichia coli and Staphylococcus aureus were tested. Among these agents obtained, compound 24 showed significantly enhanced activity against S. aureus. The results indicate that there is much room for modifications at the N-1 position.


Development, synthesis, and biological evaluation of (-)-trans-(2S,5S)-2-[3-[(2-oxopropyl)sulfonyl]-4-n-propoxy-5-(3- hydroxypropoxy)-phenyl]-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran, a potent orally active platelet-activating factor (PAF) antagonist and its water-soluble prodrug phosphate ester.

(-)-trans-(2S,5S)-2-[3-[(2-Oxopropyl)sulfonyl]-4-n-propoxy-5-(3- hydroxypropoxy)phenyl]-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran (10) is one of the most potent platelet-activating factor (PAF) antagonists in vitro and in vivo developed to date. This diaryltetrahydrofuran derivative evolved from modifications of MK 0287 which has been evaluated in clinical studies for asthma. Two structural modifications of MK 0287 were made: (1) elaboration of the 3'-[(hydroxyethyl)sulfonyl] group to a beta-keto propylsulfonyl, and (2) replacement of the 5'-methyl ether by a 3-hydroxypropyl ether. Compound 10 potently and specifically inhibits the binding of [3H]-C18-PAF to human platelet membranes (Ki 1.85 nM) and PMN membranes (Ki 2.89 nM). In vivo, 10 inhibits PAF-induced plasma extravasation and elevated N-acetyl-beta-D-glucosaminidase (NAGA) levels in male rats with ED50 values of 60 micrograms/kg, po and 4 micrograms/kg, iv respectively, and inhibits PAF-induced bronchoconstriction in guinea pigs with an ED50 value of 15 micrograms/kg after intraduodenal administration. Compound 15, a water-soluble phosphate ester prodrug derivative of 10 is at least equipotent to 10 in the in vivo models. Compound 19S, the primary and major metabolite of 10 and 15, is equipotent in in vitro and in vivo models.


Nonprostanoid prostacyclin mimetics. 2. 4,5-Diphenyloxazole derivatives.

4,5-Diphenyl-2-oxazolenonanoic acid (18b) was synthesized and found to inhibit ADP-induced aggregation of human platelets with an IC50 of 2.5 microM. Acid 18b displaced [3H]iloprost from human platelet membranes in a concentration-dependent fashion, consistent with 18b inhibiting platelet function by acting as a prostacyclin mimetic. By inserting a phenoxy ring into the side-chain moiety of 18b and systematically varying the pattern of substitution and length of the tethers, more potent inhibitors of platelet aggregation were identified. A phenoxy ring inserted centrally in the side chain proved to be the optimal arrangement but significant activity was observed when the aromatic ring was bound directly to the 2 position of the heterocycle. The meta-substituted cis-(ethenylphenoxy)acetic acid 37 is the most potent platelet aggregation inhibitor synthesized as part of this study with an IC50 of 0.18 microM. Acid 37 displaces [3H]iloprost from human platelet membranes with an IC50 of 6 nM. The trans-olefinic isomer of 37 (25p) is 72-fold weaker as an inhibitor of ADP-induced platelet aggregation, but the saturated derivative 25w (BMY 42393) is intermediate in potency. Structure-activity studies using 25w as a template focused on modification of the tethers intervening between the side-chain phenyl ring and the oxazole and carboxylate termini and substitution of the phenyl ring. These studies revealed that biological activity was sensitive to both the identity of the concatenating atoms and the pattern of ring substitution. The structure-activity relationships provide insight into the topographical relationship between the diphenylated oxazole ring and the carboxylic acid terminus that comprise the nonprostanoid prostacyclin mimetic pharmacophore.


Nonprostanoid prostacyclin mimetics. 3. Structural variations of the diphenyl heterocycle moiety.

4,5-Diphenyl-2-oxazolenonanoic acid (2) and 2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid (3) were previously identified as nonprostanoid prostacyclin (PGI2) mimetics that inhibit ADP-induced aggregation of human platelets in vitro. The effects on biological activity of substitution and structural modification of the 4- and 5-phenyl rings of 3 was examined. Potency showed a marked sensitivity to the introduction of substituents to these aromatic rings and only the bis-4-methyl derivative 9j, IC50 = 0.34 microM, demonstrated enhanced potency compared to the parent structure 3, IC50 = 1.2 microM. Substitution at the ortho or meta positions of the phenyl rings, replacement by thiopheneyl or cyclohexyl moieties, or constraining in a planar phenanthrene system resulted in compounds that were less effective inhibitors of ADP-induced platelet aggregation. In contrast, variation of the heterocycle moiety revealed a much less stringent SAR and many 5- and 6-membered heterocycles were found to effectively substitute for the oxazole ring of 2 and 3. The diphenylmethyl moiety functioned as an effective isostere for 4,5-diphenylated heterocycles since 13aad showed similar platelet inhibitory activity to 3. With the exception of the 3,4,5-triphenylpyrazole derivative 13g, compounds presenting the (m-ethylphenoxy)acetic acid side chain discovered with 3 demonstrated enhanced potency compared to the analogously substituted alkanoic acid derivative. The structure-activity findings led to a refinement of a model of the nonprostanoid PGI2 mimetic pharmacophore.


Flavones. 3. Synthesis, biological activities, and conformational analysis of isoflavone derivatives and related compounds.

A series of 2-alkylisoflavone derivatives 1 was prepared with the intent to study the importance of the phenyl group (at the 3-position) of the isoflavone in imparting antihypertensive activity and the substitution effects at the 2-position of isoflavone. With the exception of the 2-isopropyl analog, the antihypertensive activity of these compounds appears to have a slow onset and long duration. None of the analogs appears better than the corresponding flavone (3) and 3-phenylflavone (2) analogs. An unsuccessful attempt to correlate the relationship between antihypertensive activity and the calculated torsional angle of C2-C3-C1'-C2' is discussed. Antiinflammatory activities of these compounds along with 7-(oxypropylamine)flavones were also evaluated and found to be not very potent. The antiinflammatory activity appears to be sensitive to steric effects of the alkyl group on the nitrogen and of substituents at the 2-position of the isoflavones, while the hydroxyl group of the propanolamine side chain is not essential.


Renin inhibitors containing new P1-P1' dipeptide mimetics with heterocycles in P1'.

A series of renin inhibitors containing new P1-P1' dipeptide mimetics are presented. The P1-P1' mimetics were obtained from (4S,5S)-3-(tert-butoxycarbonyl)-4-(cyclohexylmethyl)-5-[(omega- mesyloxy)alkyl]-2,2-dimethyloxazolidines 5b, 9, and 11b by nucleophilic substitution of the mesylate groups with the sodium salts of mercapto- and hydroxyheterocycles. Removal of the protecting groups and stepwise acylations with amino acid derivatives provided renin inhibitors with a length of a tripeptide. Replacement of P2 histidine by other amino acids maintained or enhanced renin inhibitory potency. By alteration of P3 phenylalanine, compounds with IC50 values in the nanomolar range and stability against chymotrypsin were obtained. Finally, the effect of the C-terminal heterocycle on the renin inhibition was studied. Compound XVII was examined in vivo for its hypotensive effects. In salt-depleted cynomolgus monkeys, XVII inhibited plasma renin activity and lowered blood pressure after oral administration of a dose of 10 mg/kg.


6-Alkyl-12-formylindolo[2,1-a]isoquinolines. Syntheses, estrogen receptor binding affinities, and stereospecific cytostatic activity.

A number of 6-alkyl-12-formyl-5,6-dihydroindolol[2,1-a]isoquinolines were synthesized by the Bischler-Napieralski reaction from the respective 1-alkyl-2-(3-methoxyphenyl)ethylamines and bromo-substituted (methoxyphenyl)acetic acid chlorides followed by a second ring closure reaction involving a base-generated benzyne intermediate. The methoxy functions in positions 3 and 9 or 10 were cleaved with BBr3 and the free hydroxy groups converted into the acetates. The enantiomers of the most potent derivatives were separated by liquid chromatography on triacetylcellulose. All of the compounds tested bind to the calf uterine estrogen receptor. The relative binding affinities (RBA) ranged from 0.5 to 3.9 (17 beta-estradiol: RBA = 100) and were dependent on the position of the oxygen function in the indole moiety. The 3,10-diacetoxy derivatives showed higher RBA values than the corresponding 3,9-substituted tetracycles. There was no major difference in binding affinity between (+)- and (-)-enantiomers. Computer-assisted molecular modeling studies showed that the chiral carbon atom 6 of the indoloisoquinoline is likely to mimic the C-11 atom of estradiol. In the mouse uterine weight test, only the 3,10-diacetoxy series exhibited weak estrogenic activity at higher doses. The antiestrogenic effects found with all the compounds varied considerably. Maximum inhibition of estrone-stimulated uterine growth was found for the ethyl derivative 7d (80% with 250 micrograms/animal per day). All derivatives strongly inhibited the growth of human breast cancer cells in vitro. There was no significant difference between hormone-sensitive MCF-7 cells and hormone-independent MDA-MB 231 cells. Cytostatic activity was higher for the 3,9-substituted indoloisoquinolines than for the 3,10-analogues and dependent on the length of the alkyl group at C-6. The maximum effect was found with the butyl derivative 7g. When the enantiomers of the ethyl (7c), propyl (7e), and butyl derivative were studied, a strong difference in activity was observed between the stereoisomers. The IC50 values of the (+)-forms were usually only a tenth of those of the levorotatory isomers. Maximum cytostatic activity was found with (+)-7g: 85% inhibition at 1 x 10(-7) M in MCF-7 cells and 94% inhibition at 2.5 x 10(-7) M in MDA-MB 231 cells. This stereospecificity indicates a selective action on a biochemical target. Since no interaction with DNA was observed, the precise mode of action still remains to be elucidated.


6-substituted decahydroisoquinoline-3-carboxylic acids as potent and selective conformationally constrained NMDA receptor antagonists.

We have prepared a series of 6-substituted decahydroisoquinoline-3-carboxylic acids, and structurally similar analogs, as potential N-methyl-D-aspartate receptor antagonists. There is a large body of evidence to support the use of such compounds as cerebroprotective agents in a variety of acute and chronic neurodegenerative disorders, where some component of glutamate-mediated excitotoxicity may exist. The compounds prepared were evaluated in vitro in both receptor binding assays ([3H]CGS19755, [3H]AMPA, and [3H]kainic acid) and in a cortical wedge preparation (versus NMDA, AMPA, and kainic acid) to determine affinity, potency, and selectivity. The new amino acids were also evaluated in vivo for their ability to block NMDA-induced lethality in mice. We synthesized many of the possible diastereomers of the decahydroisoquinoline nucleus in order to examine the spatial and steric requirements for affinity at the NMDA receptor and activity as NMDA antagonists. From our structure-activity relationship we identified two potent and selective NMDA receptor antagonists, the phosphonate- and tetrazole-substituted amino acids 31a and 32a, respectively, that show good activity in animals following systemic administration. For example, 31a and 32a selectively displaced [3H]CGS19755 binding with IC50S of 55 +/- 14 and 856 +/- 136 nM, respectively, and selectively antagonized responses due to NMDA in a cortical wedge preparation with IC50S of 0.15 +/- 0.01 and 1.39 +/- 0.29 microM, respectively. And compounds 31a and 32a blocked NMDA-induced lethality in mice with minimum effective doses of 1.25 and 2.5 mg/kg (intraperitoneal), respectively. These novel amino acids are among some of the most potent NMDA antagonists described thus far, and are excellent candidates for development as neuroprotective agents for a number of CNS disorders.


5,5-disubstituted hydantoins: syntheses and anti-HIV activity.

A series of 5,5-disubstituted hydantoin derivatives was synthesized by alkylating 5,5-bis(mercaptomethyl)-2,4-imidazolidinedione (3) with various halomethylaromatic or halomethylheteroaromatic precursors, or by using the Buchener-Berg procedure on the required ketone. When evaluated for their ability to inhibit HIV-induced cell killing and virus production in CEM or MT-2 cells only compounds 2, 4n, 4o, and 4i demonstrated modest activity, the latter with an IC50 = 53 microM.


2-Nitroimidazole dual-function bioreductive drugs: studies on the effects of regioisomerism and side-chain structural modifications on differential cytotoxicity and radiosensitization by aziridinyl and oxiranyl derivatives.

A series of 2-nitroimidazoles bearing side chains terminating in or containing aziridinyl and oxiranyl groups has been prepared, and the compounds were evaluated in vitro as hypoxia-selective bioreductively-activated cytotoxins and selected compounds tested for their radiosensitizing properties toward hypoxic mammalian cells. Compounds were either the regioisomers of analogues of the potent dual-functional 2-nitroimidiazole alpha-[(1-aziridinyl)-methyl]-2-nitro-1H-imidazole-1- ethanol (RSU-1069, 1) with additional methyl groups or related oxiranes of varying side-chain length and type. Oxiranyl derivatives showed little differential toxicity, and those tested were less effective as radiosensitizers, and although these properties were influenced by side-chain length, differences were not great. Aziridinyl compounds related to 1 but with increased side-chain lengths were unstable. Methylation of 1 in various regions had little effect on radiosensitization and no clear advantages over 1 as differential cytotoxic drugs. Progressive methylation at C-3 was found to increase toxicity but decrease hypoxia selectivity. Incorporation of a cyclohexane side chain in 1,2-cis-2,3-trans-3-aziridin-1-yl-2-hydroxy-1-(2-nitroimidazol+ ++-1- yl)cyclohexane (26) abolished hypoxia-selective toxicity and unexpectedly reduced radiosensitizing efficiency. Of the aziridines, 1-(2-nitro-1-imidazolyl)-2-methyl-3-(1-aziridinyl)-2-propanol (20) was comparable in efficacy to 1 as a bioreductively-activated cytotoxin with slightly lower aerobic toxicity; however, the prodrugs of 1 remain as preferred candidates for clinical evaluation.


TCDD causes stimulation of c-ras expression in the hepatic plasma membranes in vivo and in vitro.

A series of in vivo and in vitro experiments were conducted to determine the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) administered on the expression of c-ras. Differences in c-ras expression between control and TCDD treated groups were determined by immunoassay of p21ras protein, or indirectly measured by the specific binding of 3H-GTP to hepatic plasma membrane preparations. Intraperitoneal injection of sublethal doses of TCDD significantly elevated (P less than 0.05, Student t test) levels of hepatic p21ras protein in Sprague-Dawley rats and TCDD sensitive C57BL/6J mice. Such an increase occurred at an early stage of poisoning in the C57BL/6J mice. The earliest increase was detectable 6 hr after dosing, and the difference became statistically significant by 12 and 24 hr after dosing. In contrast, TCDD tolerant DBA/2J mice had only a marginal increase in hepatic p21ras protein which did not become statistically significant even at 24 hr host-dosing. TCDD evoked increases in hepatic p21ras protein of C57BL/6J mice were accompanied by the increase in the specific binding of GTP to hepatic plasma membranes. Column chromatography of solubilized rat hepatic membrane proteins on sephadex G-50 showed TCDD administration increased levels of a 3H-GTP binding protein with MW of approximately 21 Kd. 3H-GTP binding in total hepatic membranes was also elevated (P less than 0.05, Fisher PLSD multiple comparison test) 6 hr and 24 hr after dosing of C57BL/6J mice, but as expected the effect of TCDD was not as conspicuous as that found in the plasma membrane. TCDD treatment increased levels of a 21 Kd protein found in the in vitro translation products of RNA purified from guinea pig liver. This protein was identified as a c-ras protein based upon its ability to bind GTP, precipitation by a polyclonal antibody against the rasHa and Ki proteins and subsequent SDS-PAGE which showed a single protein band of approximately 21 Kd.


Oxidant and angiotensin II-induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells.

We recently reported that nitrogen dioxide (NO2), an environmental oxidant, alters the dynamics of the plasma membrane lipid bilayer structure, resulting in increased phosphatidylserine content and angiotensin II (Ang II) receptor binding. Angiotensin II is known to elicit receptor-mediated stimulation of diacylglycerol (DAG) production in pulmonary artery endothelial cells. Because protein kinase C (PKC) is a phosphatidylserine-dependent enzyme and is activated by DAG, we examined whether NO2 resulted in activation and/or translocation of PKC from predominantly cytosolic to membrane fractions of these cells. We also evaluated whether NO2 exposure resulted in increased production of DAG in pulmonary artery endothelial cells. Exposure to 5 ppm NO2 for 1-24 hr resulted in significant increases in PKC activity in the cytosolic and membrane fractions (p less than 0.05 for both fractions) compared to activities in control fractions. Exposure to Ang II resulted in translocation of PKC activity from cytosol to membrane fractions of both control and NO2-exposed cells. This translocation of PKC from cytosolic to membrane fraction was prevented by the specific receptor antagonist [Sar1 Ile8] Ang II. Exposure of 5 ppm NO2 for 1-24 hr provoked rapid increases in [3H]glycerol labeling of DAG in pulmonary artery endothelial cells. These results demonstrate that exposure to NO2 increases the production of second messenger DAG and activates PKC in both the cytosolic and membrane fractions, whereas Ang II stimulates the redistribution of PKC from cytosolic to membrane fractions of pulmonary artery endothelial cells.


The role of calcium ions for the expression of ricin toxicity in cultured macrophages.

Ricin toxin, which consists of two distinct polypeptide moieties, A and B chains, is cytotoxic to the cultured macrophage cell line, J774A.1. Ricin is a protein synthesis inhibitor, and incubating macrophages for 4 hours with ricin (1 pM to 10 nM) in standard medium containing calcium and magnesium inhibited 3H-leucine incorporation into protein (97%, at 1 nM ricin). However, in Ca(2+)-free medium, protein synthesis was inhibited only 19%. EGTA pretreatment (to deplete intracellular calcium) also partly protected cells from protein synthesis inhibition, in spite of added calcium (2 mM) in the incubation medium. Decreased toxicity in the absence of extracellular calcium resulted from decreased toxin binding. Adding or deleting Mg2+ did not affect protein synthesis or binding of 125I-ricin in cultured macrophages. We conclude that calcium is required for ricin to exert its inhibitory effect on protein synthesis in cultured macrophages.


Modifiers of mercaptopyruvate sulfurtransferase catalyzed conversion of cyanide to thiocyanate in vitro.

The enzyme mercaptopyruvate sulfurtransferase appears to play an important role in the in vivo detoxification of cyanide. It does so by transferring sulfur to cyanide to produce thiocyanate, which is less toxic and may be excreted through the kidney. Several compounds were tested for their ability to affect the rate of enzyme catalyzed thiocyanate formation in vitro. The studies were carried out using both a partially purified bovine kidney extract and a highly purified enzyme preparation. Hypotaurine and methanesulfinic acid doubled sulfurtransferase activity in the partially purified extract at 30 mM, but inhibited the purified enzyme to 57% (hypotaurine) and 27% (methanesulfinic acid) of control activity at the same concentration. Pyruvate, phenylpyruvate, oxobutyrate, and oxoglutarate each inhibited the extract and purified forms of mercaptopyruvate sulfurtransferase. Phenylpyruvate was the most effective inhibitor, reducing activity to 0.2% of control values in the extract, and 11% of control values for purified MPST when added to the reaction at 30 mM. Other compounds tested (see Table 1) had a negligible effect on sulfurtransferase activity. A heat stable cofactor was found in boiled kidney extract which stimulated sulfurtransferase activity in the extract but inhibited sulfurtransferase activity in the purified enzyme, as was observed for hypotaurine and methanesulfinate. The boiled extract had no thiocyanate forming activity of its own. The cofactor operated in synergy with methanesulfinate, but independently of hypotaurine.


Biological action of lantadene C, a new hepatotoxicant from Lantana camara var. aculeata.

Lantadene C (22 beta-2-methylbutanoyloxy-3-oxoolean-12-en-28-oic acid) isolated from the leaves of the hepatotoxic plant Lantana camara var. aculeata (Red) has been found to be identical with dihydrolantadene A reported earlier. Molecular structure of lantadene C has been deduced from single crystal X-ray diffraction analysis. It resembles lantadene A in the pentacyclic portion of the molecule but differs in the side chain region. Atom C-34 is cis to C-35 in lantadene C but is trans in lantadene A. Semisynthetic lantadene C was prepared by catalytic hydrogenation of lantadene A. Lantadene C was obtained in two forms, I and II. Form I was crystalline while form II was amorphous. Unlike lantadene A, both form I and II of lantadene C elicited strong hepatotoxic response in guinea pigs associated with decrease in fecal output, feed intake, hepatomegaly, hepatic injury at the cellular and subcellular level, increase in plasma bilirubin, and acid phosphatase activity. All the clinical signs, hepatic lesions, and changes in blood plasma typified lantana toxicity. This is the first report on the hepatoxicity of lantadene C. The interrelation of molecular structure and biological activity of lantadene A and C has been discussed.


Whether cytochrome P450 induction accompanies glucuronosyl and glutathione transferase induction by isomers of dipyridyl appears unrelated to dose and iron chelation properties.

Administration of 4,4'dipyridyl to rats induces the activities of xenobiotic transferases (phase II drug metabolizing enzymes), UDP-glucuronosyl-transferase and glutathione-S-transferase, and also the concentration and activity of cytochrome P450 (a phase I drug metabolizing enzyme). 2,2'Dipyridyl, an isomer possessing iron chelation properties, only induces the phase II enzymes. Although the magnitude of the phase II induction by 2,2'dipyridyl increases with increasing dosages, the selective induction of only phase II activities remains inviolate. Co-administration of 2,2'dipyridyl does not prevent 4,4'dipyridyl from inducing cytochrome P450, suggesting that the iron chelation property is not the factor that precludes 2,2'dipyridyl from coordinately inducing cytochrome P450 with the transferases.


Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes.

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Light-induced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protoporphyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.