Prospective evaluation of treatment response and disease reversibility of paediatric localized scleroderma (morphoea) to steroids and methotrexate using multi-modal imaging.

BACKGROUND: Paediatric localized scleroderma is a severe inflammatory disorder associated with tissue atrophy, often leading to disability. Assessing disease activity and response to treatment has always been challenging and remains an important difficulty in clinical practice. OBJECTIVES: To investigate prospectively the efficacy of systemic treatment with corticosteroids and methotrexate in children with localized scleroderma and the validity of infrared thermography, laser Doppler flowmetry and high-frequency ultrasound in assessing disease activity. METHODS: Children with localized scleroderma were prospectively treated with corticosteroids (initially pulsed IV methylprednisolone 30 mg/kg/day, maximum 500 mg/day and/or oral prednisolone 0.5-1 mg/kg/day) and methotrexate (15 mg/m2 weekly). Treatment response was evaluated using a clinical activity score. Skin temperature, blood flow, dermal thickness and dermal echogenicity of clinically active skin lesions were determined in relation to the unaffected contralateral site at baseline and after 3, 6, 12 and 18 months. Patient charts were later reviewed for long-term follow-up. RESULTS: Twenty-two patients were included [age 6.0 (0.2-14.4] years; female-to-male ratio 3.4 : 1) All responded well to therapy. Disease reversibility was demonstrated in the majority of children with partial resolution of skin sclerosis and regrowth of hair. Laser Doppler flowmetry and high-frequency ultrasound findings correlated with disease activity at baseline. Thermography had no added value in this cohort. The recurrence rate was 36% in the follow-up period. CONCLUSIONS: Corticosteroids and methotrexate are highly effective as first-line therapy in paediatric localized scleroderma, leading to partial reversal of skin manifestations. However, the recurrence rate is substantial and affected children require long-term follow-up. Laser Doppler flowmetry and high-frequency ultrasound correlate with disease activity in the acute phase and may assist decision-making in these patients.

Classification of neurological abnormalities in children with congenital melanocytic naevus syndrome identifies magnetic resonance imaging as the best predictor of clinical outcome.

BACKGROUND: The spectrum of central nervous system (CNS) abnormalities described in association with congenital melanocytic naevi (CMN) includes congenital, acquired, melanotic and nonmelanotic pathology. Historically, symptomatic CNS abnormalities were considered to carry a poor prognosis, although studies from large centres have suggested a much wider variation in outcome. OBJECTIVES: To establish whether routine MRI of the CNS is a clinically relevant investigation in children with multiple CMN (more than one at birth), and to subclassify radiological abnormalities. METHODS: Of 376 patients seen between 1991 and 2013, 289 fulfilled our criterion for a single screening CNS MRI, which since 2008 has been more than one CMN at birth, independent of size and site of the largest naevus. Cutaneous phenotyping and radiological variables were combined in a multiple regression model of long-term outcome measures (abnormal neurodevelopment, seizures, requirement for neurosurgery). RESULTS: Twenty-one per cent of children with multiple CMN had an abnormal MRI. Abnormal MRI was the most significant predictor of all outcome measures. Abnormalities were subclassified into group 1 'intraparenchymal melanosis alone' (n = 28) and group 2 'all other pathology' (n = 18). Group 1 was not associated with malignancy or death during the study period, even when symptomatic with seizures or developmental delay, whereas group 2 showed a much more complex picture, requiring individual assessment. CONCLUSIONS: For screening for congenital neurological lesions a single MRI in multiple CMN is a clinically relevant strategy. Any child with a stepwise change in neurological/developmental symptoms or signs should have an MRI with contrast of the brain and spine to look for new CNS melanoma.

Growth and hormone profiling in children with congenital melanocytic naevi.

BACKGROUND: Multiple congenital melanocytic naevi (CMN) is a rare mosaic RASopathy, caused by postzygotic activating mutations in NRAS. Growth and hormonal disturbances are described in germline RASopathies, but growth and hormone status have not previously been investigated in individuals with CMN. OBJECTIVES: To explore premature thelarche, undescended testes, and a clinically abnormal fat distribution with CMN through prospective endocrinological assessment of a cohort of subjects with CMN, and a retrospective review of longitudinal growth of a larger group of patients with CMN from outpatient clinics (which included all subjects in the endocrinological assessment group). PATIENTS AND METHODS: Longitudinal growth in a cohort of 202 patients with single or multiple CMN was compared with the U.K. National Child Measurement Programme 2010. Forty-seven children had hormonal profiling including measurement of circulating luteinizing hormone, follicle-stimulating hormone, thyroid stimulating hormone, adrenocorticotrophic hormone, growth hormone, prolactin, pro-opiomelanocortin, estradiol, testosterone, cortisol, thyroxine, insulin-like growth factor-1 and leptin; 10 had oral glucose tolerance testing 25 had dual-energy X-ray absorptiometry scans for body composition. RESULTS: Body mass index increased markedly with age (coefficient 0·119, SE 0·016 standard deviation scores per year), at twice the rate of the U.K. population, due to increased adiposity. Three per cent of girls had premature thelarche variant and 6% of boys had persistent undescended testes. Both fat and muscle mass were reduced in areas underlying large naevi, resulting in limb asymmetry and abnormal truncal fat distribution. Anterior pituitary hormone profiling revealed subtle and variable abnormalities. Oral glucose tolerance tests revealed moderate-severe insulin insensitivity in five of 10, and impaired glucose tolerance in one. CONCLUSIONS: Interpersonal variation may reflect the mosaic nature of this disease and patients should be considered individually. Postnatal weight gain is potentially related to the underlying genetic defect; however, environmental reasons cannot be excluded. Naevus-related reduction of fat and muscle mass suggests local hormonal or metabolic effects on development or growth of adjacent tissues, or mosaic involvement of these tissues at the genetic level. Premature thelarche and undescended testes should be looked for, and investigated, as for any child.

New vascular classification of port-wine stains: improving prediction of Sturge-Weber risk.

BACKGROUND: Facial port-wine stains (PWSs) are usually isolated findings; however, when associated with cerebral and ocular vascular malformations they form part of the classical triad of Sturge-Weber syndrome (SWS). OBJECTIVES: To evaluate the associations between the phenotype of facial PWS and the diagnosis of SWS in a cohort with a high rate of SWS. METHODS: Records were reviewed of all 192 children with a facial PWS seen in 2011-13. Adverse outcome measures were clinical (seizures, abnormal neurodevelopment, glaucoma) and radiological [abnormal magnetic resonance imaging (MRI)], modelled by multivariate logistic regression. RESULTS: The best predictor of adverse outcomes was a PWS involving any part of the forehead, delineated at its inferior border by a line joining the outer canthus of the eye to the top of the ear, and including the upper eyelid. This involves all three divisions of the trigeminal nerve, but corresponds well to the embryonic vascular development of the face. Bilateral distribution was not an independently significant phenotypic feature. Abnormal MRI was a better predictor of all clinical adverse outcome measures than PWS distribution; however, for practical reasons guidelines based on clinical phenotype are proposed. CONCLUSIONS: Facial PWS distribution appears to follow the embryonic vasculature of the face, rather than the trigeminal nerve. We propose that children with a PWS on any part of the 'forehead' should have an urgent ophthalmology review and a brain MRI. A prospective study has been established to test the validity of these guidelines.

Proteins associated with the early intrauterine equine conceptus.

A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16-17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F(2 alpha) (PGF(2 alpha)). Uterocalin and beta(2)-microglobulin (beta(2)M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were similar in conceptuses from control and treated mares. A 17 kDa cationic protein identified as a secretory phospholipase A2 (sPLA2) type IIA was detected bound to normal capsules but increased substantially in response to PGF(2 alpha). Two forms of uteroglobin were distinguished by partial amino acid sequences of approximately 6 kDa bands in flush fluids from normal pregnant uteri. After administration of PGF(2 alpha) one immunoreactive form of uteroglobin was preferentially increased. These studies demonstrate that failure of pregnancy in this model is associated with an increase in secretory phospholipase in the capsule and a change in the forms of uteroglobin in the uterine secretions.

Stimulation of cell proliferation by calcium and a calcimimetic compound.

Some mesenchymal cells respond to stimulation by specific cations with increased cell proliferation. In the present study we have investigated whether the parathyroid/kidney/brain calcium-sensing receptor (PCaR) can mediate such mitogenic responses. We have expressed the recombinant rat PCaR in CCL39 hamster fibroblasts, which do not express a detectable endogenous cation sensor. The transfected cells responded to increased extracellular calcium concentrations ([Ca2+]e) with strong inositol phosphate (IP) formation, which was insensitive to pertussis toxin treatment of cells. We could not detect negative coupling of the receptor to adenylyl cyclase. The calcimimetic NPS R-568 left-shifted the concentration-response curve for [Ca2+]e-induced IP formation and increased the maximal response. In [3H]thymidine incorporation experiments, increasing [Ca2+]e from 1 to 4 mM was found to stimulate DNA synthesis weakly, but significantly. A strong potentiation of this response was observed in the presence of NPS R-568. [Ca2+]e and NPS R-568 also synergized to increase cell numbers in cultures maintained in defined medium. In contrast to our expectations, no significant stimulation of IP formation or cell proliferation could be observed after stimulation of cells with the reported PCaR agonist gadolinium (Gd3+) or with aluminum (Al3+), which stimulates osteoblast proliferation. Gd3+ actually inhibited IP formation stimulated by increased [Ca2+]e as well as by thrombin and AlF4-, indicating toxicity. However, submaximal receptor stimulation by Gd3+ was evident when intracellular calcium transients were measured in fluo-3-loaded cells. Our data show that PCaR can stimulate cell proliferation when expressed in an appropriate cellular context. However, it is unlikely that PCaR mediates the strong mitogenic effects elicited by the cations Gd3+ and Al3+ observed in osteoblasts.

Osmolality of equine blastocyst fluid from day 11 to day 25 of pregnancy.

Horse conceptuses collected between Day 11 and Day 18 of pregnancy float in isotonic media. To investigate this phenomenon, blastocyst fluids from 30 conceptuses from 13 mares were analysed for osmolality and for concentrations of Na+, Cl-, K+, glucose, urea and creatinine. In conceptuses from Group A, samples from Day 11 to Day 16 yielded the following results (mean +/- s.e.m.): osmolality, 121.4 +/- 1.5 mOsm kg-1; Na+, 11.0 +/- 2.2 mM; Cl-, 29.3 +/- 2.5 mM; K+, 26.2 +/- 2.6 mM; glucose, 0.6 +/- 0.1 mM; urea, 6.0 +/- 0.6 mM; creatinine, 9.6 +/- 1.1 microM. Between Day 16 and Day 25, the osmolalities and Na+ concentrations increased gradually with age but the former never exceeded 255 mOsm kg-1. Fluids from Group B were obtained from eight conceptuses exposed to saline solutions of different osmolalities for various periods of time. An increased perivitelline space of 1-3 mm became evident at the lower pole of the floating conceptus after 45 min of exposure to solutions with osmolalities of > or = 300 mOsm kg-1, suggesting that Na+ and Cl- diffuse freely through the capsule but not through the trophoblast. The significance of the hypo-osmolality of equine blastocyst fluid is discussed but remains unclear.

Immunohistochemical evaluation of the equine endometrium during the oestrous cycle.

Endometrial biopsies were obtained from four mares during consecutive oestrous cycles on the first day of oestrus, on the day when ovulation was detected, and four and eight days after ovulation. Cycle stages were confirmed by means of rectal palpation, ultrasonography and plasma progesterone determination. Immunohistochemical evaluation of the formalin fixed biopsy specimens was performed using a peroxidase anti-peroxidase technique. Immunoglobulin (Ig)A-, IgM-, IgG(Fc)- and IgG(T)-containing cells were detected in all biopsies; with IgA- and IgG(Fc)-containing cells generally predominating. There was no cyclical trend of Ig-containing cell numbers for any isotype. Free immunoglobulins of the four classes evaluated were frequently seen in luminal epithelium, glandular epithelium and secretions, and interstitium. This study of endometrial biopsies from a limited number of cycling mares suggests the presence in the equine endometrium of free and intracellular immunoglobulins of the classes A, M, G(Fc) and G(T) without any apparent cyclical trend.

Mononuclear cell infiltration of the equine endometrium: immunohistochemical studies.

Endometrial sections from mares with varying degrees of mononuclear cell infiltration were examined for immunoglobulin (Ig)A-, IgM-, IgG(T)- and IgG(Fc)-containing cells, luminal and glandular epithelial cell Ig-staining and free interstitial Ig-staining, using a peroxidase anti-peroxidase technique. Mares with mild to moderate (Group 2) and mares with severe diffuse mononuclear cell infiltration, superimposed by acute endometritis (Group 3), had significantly higher numbers of Ig-containing cells than genitally-normal mares (Group 1). The differences between Groups 1 and 3 were significant for all four isotypes. In Groups 1 and 2, numbers of IgA-containing cells were significantly larger than numbers of IgM- and IgG(T)-containing cells. Generally, more glandular epithelial cells stained for IgA and IgM than for IgG(T) and IgG(Fc), and Ig-staining for all isotypes increased from Group 1 to Group 3. Free interstitial staining did not appear to differ among the three groups, but IgG(Fc)- and IgG(T)-staining generally was more intense than IgA- and IgM-staining. The efficiency of uterine defence in the mare does not seem to depend solely on humoral factors, and defects involving other components of the defence system may contribute to failure of the uterus to clear infection.

Relationships of total protein, specific gravity, viscosity, refractive index and latex agglutination to immunoglobulin G concentration in mare colostrum.

A colostrum sample was collected within 24 h after foaling from 27 mares and from 10 other mares a milk sample was collected several weeks post partum. Immunoglobulin G concentrations were determined quantitatively by radial immunodiffusion and semi-quantitatively using a commercial latex agglutination test. Total protein, specific gravity, viscosity and refractive index were determined and their relationships to the immunoglobulin G concentration analysed. All parameters correlated with the immunoglobulin G concentration. The latex agglutination test divided the colostrum samples into three groups with different means for immunoglobulin G and total protein concentrations. Specific gravity and the latex agglutination test were found to be the methods best suited for on-farm evaluation of colostrum quality.

Streptomycin-resistant Escherichia coli as a marker of vulvovestibular contamination of endometrial culture swabs in the mare.

To investigate the vulvovestibular contamination of endometrial culture swabs in the mare, a liquid culture of a streptomycin-resistant strain of Escherichia coli was applied to the vulvovestibular area of mares and used as a marker of contamination of endometrial culture swabs. Prior to taking endometrial swabs, the perineal area was washed with soap, rinsed with water, and dried. Endometrial culture swabs were taken from mares that were in anestrus or diestrus and from mares that were in estrus. When a manual transvaginal swabbing technique was used, 22 of 24 endometrial swab specimens from 12 mares were contaminated with the experimental bacterial strain; culture of only one endometrial swab yielded more than nine colonies. When a speculum approach was employed, three of 12 swab specimens from 12 mares yielded between one and three colonies. The stage of cycle had no effect on the extent of contamination, but the proportion of positive cultures was significantly smaller when swabs were taken via a vaginal speculum approach, compared to a manual transvaginal approach. Complete preclusion of vulvovestibular contamination of endometrial swab specimens was not achieved; however, fewer than ten colonies can be expected even in mares in which the vulvovestibular area has been thoroughly contaminated with a broth culture, provided that the perineal area is adequately cleaned prior to swabbing.

Two related cases of cerebellar abnormality in equine fetuses associated with hydrops of fetal membranes.

Hydrops allantois was diagnosed in two Haflinger mares with severe abdominal distension. Both mares were seven months pregnant. Abortion was induced with two injections of prostaglandin six hours apart followed by further manual dilation of the cervix and administration of oxytocin the next day. There were 90 and 95 litres of fluid, respectively, in the allantoic cavities which resembled extracellular fluid with regard to concentrations of urea, creatinine, sodium, potassium, calcium, magnesium, phosphate and chloride, but not total protein. Both fetuses had severe brain abnormalities which were diagnosed as cerebellar and cerebral hypoplasia associated with bilateral hydrocephalus internus and hydranencephaly and cerebellar aplasia, respectively. Both mares were pregnant by the same stallion, but a clear hereditary link was not found.

A clinical trial on the effect of prostaglandin F2 alpha on placental expulsion in dairy cattle after caesarean operation.

One hundred and thirty dairy cattle that had undergone a caesarean operation because of dystocia were assigned either to a treatment group, receiving 25 mg dinoprost (synthetic prostaglandin F2 alpha) or to a control group, receiving 5 ml saline solution, administered intramuscularly after removal of the calf and reposition of the closed uterus into the abdominal cavity. In 52 of 65 (80.0 per cent) prostaglandin-treated animals and in 38 of 65 (58.5 per cent) control animals the complete placenta was expelled within 12 hours. This difference was statistically significant (P < 0.05).

Distribution of histological lesions in the equine endometrium.

The distribution of histopathological lesions in the equine endometrium was examined to investigate the representativeness of a single biopsy specimen in terms of the whole endometrium. Five sections from each of 110 uteri obtained from slaughtered mares were evaluated microscopically and classified according to a four-category grading system used for endometrial biopsies. Depending on the extent of agreement between the categories of the homologous sections, the uteri were considered to show either good agreement (81 uteri; 73.6 per cent), moderate agreement (26 uteri; 23.6 per cent) or poor agreement (three uteri; 2.7 per cent). All the homologous sections of the group showing moderate agreement were within two adjacent categories. Disagreements were more often due to variations in the distribution of fibrotic lesions than to variations in the degree of chronic infiltrative lesions. There was no seasonal effect on the apparent degree or distribution of lesions. In most cases the examination of a single biopsy, when combined with a thorough clinical examination, should provide adequate information about the condition of a mare's endometrium.

Endometrial biopsy in mares under nonuniform breeding management conditions: Prognostic value and relationship with age.

Endometrial biopsy plays an important role in fertility examination of the mare. The purpose of this study was to examine the prognostic usefulness of endometrial biopsy under nonuniform breeding management conditions, and to clarify any relationship with age of the mare. Based on endometrial histological changes, 192 mares from different farms were classified using a four-category grading system. There were significant associations between categories and fertility. Mean pregnancy rates in the four categories were 79, 49, 33 and 0% and mean foaling rates were 70, 42, 18 and 0%, respectively. Mares older than the median age (=11 years) had, on average, higher category scores and significantly lower fertility than younger mares. Within categories, there was a tendency towards higher fertility in younger mares. Although the precise prediction of fertility of the individual mare remains difficult, it is evident from this investigation that endometrial biopsy has considerable prognostic potential even under nonuniform breeding management conditions. Furthermore, the age of a mare must be considered when estimating her fertility potential.

[Clinico-chemical blood parameters in foals in the first two months of life]. / Klinisch-chemische Blutparameter beim Fohlen in den ersten zwei Lebensmonaten.

Eighteen healthy foals were studied from birth until 2 months of age. Blood samples were obtained at the following times: presuckle, 30 hours, 1, 3, 5, 7 and 9 weeks of age. Changes in serum P, Mg, Ca, Na, K, Cl, iron, AP, ASAT, ALAT, GGT, GLDH, CK, lipase, urea, creatinine, cholesterol, triglyceride, uric acid, protein and fibrinogen and in plasma total solids were examined and the values compared to reference values of adult horses. There were characteristic age related changes in several parameters. Single measurements should be interpreted cautiously to allow for individual variations.

[Hematological reference values for foals in the first two months of life]. / Hämatologische Referenzwerte beim Fohlen in den ersten zwei Lebensmonaten.

Hematologic reference values were established in 18 healthy foals in the first two months of life. Blood samples were collected prior to colostrum consumption and at 30 hours, 1 week, 3, 5, 7 and 9 weeks of age. PCV, Hb and RBC decreased during the first week and RBC, but not PCV and Hb, increased toward the end of the second month. With the exception of the sample at 1 week, the foals had mean RBC values significantly higher than those of controls. Mean MCV and MCH did not change during the first week, but decreased slightly thereafter; all means were smaller than in controls. The numbers of neutrophils increased mainly during the first week, and the numbers of lymphocytes increased gradually during the first two months. The mean N:L-ratio was highest at 1 week and lowest at 5 weeks.