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Pregnancy diagnosis by monitoring ovarian physiology is of particular importance in this respect cheap prednisolone 10 mg on line, particularly as a management tool for the captive Breeding Programme prednisolone 20 mg visa. In lynx purchase prednisolone 40mg fast delivery, however, fecal steroids T do not follow the typical pregnancy pattern of felids (Brown et al. Alternatively to fecal hormone metabolites, urine has been often used for tracking sexual hormones. Steroid hormones are secreted via the kidney into urine, mainly conjugated as sulfates or glucuronides. In the domestic cat, relaxin increases at the beginning of the second trimester (day 20 to 25) reaching a plateau and a subsequent decrease 10 to 15 days before parturition (Stewart and Stabenfeldt, 1985). Furthermore, a bench top kit (Witness Relaxin) was successfully used for pregnancy detection in urine collected from domestic cats (Haas van Dorsser et al. Since urine collection from captive Iberian lynx was established to supply urine for camera trapping and identifcation of free-ranging lynxes and it is performed on a regular basis at the Ex situ Breeding Programme, the aim of the present study was to test whether urine could be effectively used for pregnancy diagnosis. Our study also focused on looking for a relaxin signal in serum obtained using a minimally invasive method that involved the use of triatomine, blook- sucking, insects (Voigt et al. This study took place at the El Acebuche captive Breeding center in Doana national Park, in Southern Spain. The captive population consisted of 16 animals (seven males, nine females) in 2006 and of 52 (28 males, 24 females) in 2008 (Vargas et al. The frst birth in captivity was in 2005, since then 24 cubs have been raised in the Breeding Programme. Animals were kept in separate enclosures (550 m ), and during mating season (January to February) all females were allowed 2 to mate by introducing a male into the females enclosure. Depending upon the females behavior, the couples were maintained together for several weeks or just for the copulatory period. Mating was documented in all females, and the cycle was considered to involve a pseudo-pregnancy if no parturition or abortion was observed (Gritz et al. Prior to the test, Iberian lynx females were encouraged to use cork plates as resting spots. After 30 min of uninterrupted resting (generally sleeping) on the plates, females were removed from the enclosure and bugs were taken out of the cork holes. If samples were drawn carefully, bugs could be reutilized for other purposes, although never to draw blood again from another potentially pregnant Iberian lynx female. Blood was centrifuged to obtain plasma, a few drops of which were used for pregnancy diagnosis via Witness Relaxin, while the remaining sample was sent for additional hormone and antibody analyses. Co r k p l at e s, w h i C h ly n x e s u s e as r e s t i n g s p o t s, are equipped w i t h h o l e s Fo r speCial C o n ta i n e r s C o n ta i n i n g b l o o d -s u C k l i n g b u g s (arrow). Co m o pa r t e de la r u t i n a diaria, l o s Cuidadore s manejan l o s C o r C h o s pa r a q u e l o s l i n C e s n o sospeChen en el m o m e n t o en el q u e F i n a l m e n t e se i n t r o d u C e n l o s i n s eC to s. The collectors consisted of vertical stainless steel plates (60 x 60 cm) ending in gutters at the bottom and a slight v-shape inclination that allowed the urine to run into a collector cup. During the breeding season (January to April), samples were obtained 2-3 times per week, for the rest of the year at least one sample per month per animal was used. For steroid analysis, urine aliquots of 100 L were hydrolysed, extracted and subjected to hormone assays Fi g u r e 4. Co l l e C t i o n o F u r i n e s a m p l e s F r o m F e m a l e ly n x a t t h e el aCebuChe ib e r i a n ly n x Ca p t i v e br e e d i n g Center d u r i n g b r e e d i n g (ja n ua ry -ap r i l ) a n d n o n -b r e e d i n g (ma y -deCember) s e a s o n s 2006 to 2008 (jewgenow et a l. Urinary hormone concentrations are expressed as ng per mg creatinine in order to control for differences in urine concentration. The cross-reactivitys of both antibodies and their inter- and intra-assays coeffcients were as described before (Braun et al. The cross-reactivities of antibodies and inter- and intra-assay coeffcients were as described before (Jewgenow et al. Finally, urine samples were concentrated up to ninety times of the original urine volume. In brief, two drops of plasma were transferred to the sample well followed by two drops of the provided buffer. The appearance of a specifc relaxin and a control band was judged as an indication of relaxin in blood serum. A missing relaxin band after a prolonged time (1 hour) was an indication of a negative test result. Thus, urine was mixed with equal amounts of lynx blood serum (serum from a non-pregnant Iberian lynx female obtained during chemical immobilisation in november 2006) before it was subjected to the Witness Relaxin test. In case of concentrated urine samples, they were diluted with of blood serum from a non-pregnant Eurasian lynx and 64 L of the urine- serum mixture (estimated volume of two normal blood drops) was added to the sample well followed by two drops of the provided buffer. Positive relaxin signals were seen in the reading window within at least 20 minutes after application; control bands were visible in every test. Re s u l t s a n d d I s c u s s I o n Urinary P4 levels did not reveal a distinct increase during pregnancy compared to non-breeding season levels (Table 2). Also, no difference was found in progestin concentrations when non-pregnant (no mating), pregnant 383 Immune reactive steroid outSi de br e edi ng S e a Son Pr eg nancy un Pai r e d t teSt w i t h welch ng per mg creatinine (day 1 to 64) co rr ect ion (number of samples) MeanS ( SeM) MeanS ( SeM) Progesterone (n) 1. Co m o ejemplo s e m u e s t r a n l o s p a t r o n e s estaCionales d e d o s h e m b r a s a d u lta s sa l i e g a (p a n e l s u p e r i o r ) y au r a (p a n e l inFerior). The mean progesterone concentrations were slightly lower, when no mating occurred (Table 3, Artemisa), but progesterone levels of pseudo-pregnant and pregnant cycles did not differ signifcantly. Thus, urinary progesterone may indicate the existence of corpora lutea after induced ovulation, but a reliable pregnancy diagnosis based on progesterone was unattainable. In this respect, urinary progesterone follows the pattern described for fecal progestagen excretion in Iberian lynx females (Pelican et al. We suggest that this might be the consequence of the prolonged presence (and function) of corpora lutea throughout most of the year (Kvam, 1990; Gritz et al. Thus, a permanent progestin production may ensure the strong seasonality in lynx (but masks any changes associated with the luteal phase of pregnancy). In most felids, increased estradiol excretion is associated with estrus behaviour or exogenous gonadotropin treatment (Brown et al. Unfortunately, the collection of urine samples from individual females during the period of mating was impossible, since the husbandry scheme allows breeding pairs to share enclosures until two weeks before parturition. Urine samples collected from females with males were characterized by several-fold higher (t=35. The elevated estrogen concentrations in mixed male and female urine were found not only during co-housed pairs immediately before and during the mating period (and thus possibly related to estrus of the female), but was also seen when the couples were left together during pregnancy (three females in 2007, Table 1; Artemisa, Table 3). Within the Iberian Lynx captive Breeding Programme the collection of samples to monitor the onset of estrus in the absence of males was not possible, since all females were sharing enclosures with males prior to entering estrus. Yet, such information would be valuable for further characterization and for better understanding the onset of the Iberian lynx breeding season. The results from the female Artemisa (Table 3) showed a signifcant difference (P<0. Unfortunately, during her pregnancy in 2007, this female was kept together with a male and data on urinary estrogens (46. Although samples from only one female that failed to copulate were available, the low estrogen levels in Artemisa during the 2008 breeding season are an indication of the missing ovulation and corpora lutea (cL) formation. We conclude that Iberian lynx are induced ovulators as described for many other felid species (Brown and Wildt, 1997). Thus, if mating occurs, corpus luteum (cl) formation is evident by elevated estrogens and ultrasound examination (Gritz et al. The positively tested animals were all between day 32 and 56 of pregnancy, of a 63-66 day gestation period (Vargas et al. The animal whose plasma yielded negative WitnessRelaxin test results, was at day 32 after her frst mating, although later she was proven to be pregnant. Only when urine samples were concentrated by ultrafltration at least 50x, a weak relaxin positive reaction was observed between day 29 and 46 of the pregnancy (n=17). These failed pregnancy diagnosis might be explained by the ongoing degradation of relaxin within the urine samples (late collection) and/or insuffcient level of the ultrafltration procedure. Thus, the urinary relaxin was still under the detection level of WitnessRelaxin test. In contrast to urinary progestagens, estrogens refect cl formation after ovulation. Furthermore, the observed increase of urinary E2 after mating and during late pregnancy suggest either an E2 secretion from the lynx placenta and/or a pregnancy-specifc enhanced luteal secretion of estrogen, a point for additional study. In addition, pregnancy diagnosis is attainable by applying the Witness Relaxin bench top test, if blood plasma or highly concentrated urine samples are used. Relaxin can be detected in a certain period during pregnancy, which ranges between 34 to 50 days after mating for blood plasma, and day 37 to 46 post-mating days for urine samples, respectively.

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Single amino acid substitutions can aect proteasomal cleavage pat- terns(references in Beekman et al generic 5mg prednisolone. Instead cheap 10mg prednisolone mastercard, varying sites aect rates of cleavage and consequently relative abun- dances of dierent peptides purchase prednisolone 10mg amex. In this case, an amino acid substitution at the residue anking the C-terminus of the epitope aected both cleav- age and transport. But no data show how commonly amino acid substitutions ab- rogate ecient cleavage and transport. Experimental evolution studies could manipulate immunodominance andkinetic aspects of within-host infections to measure the frequency of the escape mechanism under dif- ferent conditions. The rather ex- treme immunodominance of this experimental system provides a good model for studying molecular details of escape variants. They isolated viruses from this later period to de- termine if escape variants had evolved and, if so, by what mechanism. Substitutions at nonanchor residues usually have much smaller eects on binding anity. They found that the peptide residue at position three had its side chain buried in the Db binding cleft and, apparently, certain substitutions such as VAat this location can disrupt binding in the manner of an anchor position (Puglielli et al. Thenine amino acids of the epitope in positions 3341 of the protein are labeled as P1P9. Three of these substitutions occurred at position 8, the primary anchor site, and one substitution occurred at position 2, the secondary anchor site. Two other substitutions reduced binding by less than two orders of magnitude: a substitutionatposition 1 reduced binding by 67%, and a substitution at position 5 reduced binding by 85%. Hosts A and D progressed slowly to disease, whereas host C progressed at an intermediate rate. The other slow progressor, host D, had all four class I molecules listed for hosts A and C, and presented all ve epitopes. For example, host C viruses were dominated by escape mutants in Env497 504 and Nef165173 but not in the other three epitopes. Ideally, experimental studies of escape would provide information about changed functional character- istics of pathogen proteins and the associated tness consequences. The Tax protein is a trans-acting transcriptional regulator that modulates expression of several viral and cellular genes (Yoshida 2001). Tax appears to aect several aspects of the cell cycle, potentially enhancing cell division and reducing cell death. Three substitutions had lowered ability to activate the viral promoter, and all nine substitutions caused lowered or no activation of two cellular promoters. Amino acid sequences of viral proteins may be shaped by two opposing pressures: contribution to viral function and escape from im- mune recognition. Thus, amino acid substitutions in response to a third force, such as a drug, may be likely to reduce protein performance or enhance recognition by the host immune system. Experimentally applied selective pressures such as drugs may provide information about the functional andimmune selective pressures that shaped the wild-type sequence. However, escape at multiple epitopes may be observed within individual hosts (Evans etal. Escapeatadominant epitope provides ben- et if the aggregate rate of killing via subdominant epitopes allows a higher probability of burst before death. If some infected cells survive to produce new virions, the benet of escape at one epitope depends on the expected increase in cellular longevity during the productive phase of virion release and the probability that released viruses transmit to new host cells. The escape mutant benets only to the extent that fewer recognized peptides occur on the cell surfacelower density may reduce the rate of killing, and that reduction may in turn allow more of the escape variants progeny to be transmitted. Higher dose most likely produces larger population size during the initial viremia, increasing the time and the number of pathogens available to make a particular mutant. Experimental manipulations could test the contributions of dosage, pathogen population size within the host, and time to clearance. Wait- ing time for an escape mutant also depends on the mutation rate, which could perhaps be varied by comparinggenotypes that diered in muta- tion rate. If the infection clears rapidly, then the potential escape variants do not increase suciently within the host to contribute signicantly to transmission to other hosts. The changing frequencies of amino acid substitutions could be tracked under dierent regimes of uctuating selection. The role of timing could be studied in the following experimental evolution design. The relative escape rates in the monoclonal hosts focused on early and late epitopes calibrate es- cape rates in the absence of competition between epitopes. In experimental evolution studies, hosts that can eectively present a broader variety of epitopes should restrict the spread of escape substitutions relative to hosts with narrower presentation. If a host is rst exposed to epitope A, subsequent exposure to epitope B tends to reinforce the response against epitope A. For example, what sort of evolutionary response would occur in a series of hosts each previously exposed to epitope A? Experimental evolutioncreatesadaptations to the particular in vitro or in vivo laboratory conditions. Laboratory studies provide an opportunity to relate biochemical mechanism to kinetics, and kinetics to tness. Mathematical models aid the controlled, experi- mental dissection of these relations (Nowak and May 2000). Controlled analysis must be complemented by study of variation and adaptation in natural isolates. Measuring Selection with Population Samples 15 Experimental evolution provides insight into kinetic and mechanistic as- pects of parasite escape from host immunity. Suchexperimental studies clarify selective forces that inuence change at certain amino acid sites. But experimental studies provide only a hint of what actually occurs in natural populations, in which selective pressures and evolutionary dy- namics dier signicantly from those in controlled laboratory studies. It is important to combine experimental insights with analyses of vari- ation in natural populations. In this chapter, I discuss how population samples of nucleotide sequences provide information about natural se- lection of antigenic variation. Ifocuson themes directly related to the goal of this bookthe syn- thesis between dierent kinds of biological analyses. In particular, I show how analysis of population samples complements studies of mo- lecular structure and experimental evolution. Several books and articles review the methods to analyze population samples and the many dier- ent types of applications (Kimura 1983; Nei 1987; Nee et al. The rst section describes how dierent kinds of natural selection cause dierent patterns of nucleotide substitutions. Thus, the pattern of nucleotide substitutions observed in a population sample of sequences can sometimes be used to infer the kind of selection. The simplest pat- tern concerns the number of nucleotide changes that cause an amino acid substitution (nonsynonymous) relative to the number of nucleotide changes that do not cause an amino acid substitution (synonymous). If natural selection does not aect the relative success of amino acid vari- ants, then nonsynonymous and synonymous nucleotide substitutions occur at the same rate. An excess of nonsynonymous substitutions sug- gests that natural selection favored those changes, providing evidence for positive selection of amino acid replacements. The second section presents two examples of positive selection on parasite antigens. Asampleofnucleotide sequences showed that strong positive selection occurred in a few small regions of the Tams1 antigen, suggesting that those regions have been under strong selection for escape from host immunity. In a sample of 892 nucleotide sequences, 77 of 86 nucleotide changes caused amino acid substitutions, a large excess of nonsynonymous substitutions. Very strong natural selection by host antibodies apparently drives rapid change in Sic. The third section continues with more examplesofpositive selec- tion on parasite antigens.

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This may be a safe and worthwhile strategy in generalized morphea cheap prednisolone 40mg amex, but requires clinical studies discount 40mg prednisolone with mastercard. Other therapies The description of a large number of anecdotal therapies for morphea attests order prednisolone 20mg with visa, in part, to their lack of efcacy. Phenytoin (200 mg /day) resulted in skin sofening within 23 months in 5 / 5 patients with linear disease (Neldner, 1978). Interestingly these patients all received concomitant vitamin D supplements, which may have contributed to the observed efects. Beneft was seen within 4 weeks of starting cyclosporine therapy (5mg/kg/day ta- pered to 1. It has also been used as an inductor treatment followed by methotrexate as maintenance treatment in a girl with linear morphea (Crespo et al. Some improvement has also been seen afer 3 months of intravenous immunoglobulin therapy (5 g alpha-globulin / day 5 days, then monthly for 1 year) in a child with pansclerotic morphea (Wollina et al. Acitretin has been used with success in 9 pa- tients (Neuhofer and Fritsch, 1984) one of them a patient with psoriasis and morphea (Bilen et al. A single report suggests the efcacy of repeated treatment with a 585 nm pulsed dye laser (Eisen and Alster, 2002) but a case reported in 2009 of a patient with en coup de saber who was given an initial diagnosis of acquired port-wine stain and treated with pulsed dye laser presented with un- anticipated blistering because of the use of the laser (Kakimoto et al. Penicillin, used because of the possible association with borrelia infection, and a suggested efect on collagen 160 Cristin Vera Kellet, Catherine H. Dutz fbrillogenesis was also recently shown to reduce skin thickness in a child with linear dis- ease (Mohrenschlager et al. It has also been used in thirteen patients with morphea without occlusion for 4 months, showing that thick, well-established lesions responded more poorly in com- parison to other less thick and more erythematous ones (Stefanaki et al. Tere is one randomized, double-blind, emollient-controlled pilot study performed in 10 patients with 4 plaque morphea showing reduction in durometer and clinical feature scores (Krof et al. Imiquimod cream 5% may have been of beneft in 15 /15 reported cases (Campione et al. Mycofenolate mofetil has been used in association with extracorporeal photopheresis with success in a patient with recalcitrant generalized bullous morphea (Schlaak et al. Abnormalities in immune system parameters, endothelial activation and fbroblast metabolism have been described, but a unifying pathophysiologic model remains to be tested. Given the benign natu- ral progression of plaque type morphea, treatment with topical modalities such as super- potent corticosteroids or calcipotriol is prudent. Recent studies support the use of methotrexate with systemic corti- costeroids for the management of aggressive disease. Resolution of therapeu- tic uncertainty must await the organization and completion of controlled trials. References Aberer E, Neumann R, Stanek G (1985) Is localised scleroderma a borrelia infection? Lan- cet 2 (8449):278 Aiba S, Tabata N, Ohtani H, Tagami H (1994) Cd34+ spindle-shaped cells selectively disappear from the skin lesion of scleroderma. Arch Dermatol 130 (5):593597 Akimoto S, Hayashi H, Ishikawa H (1992) Disaccharide analysis of the skin glycosaminoglycans in systemic sclerosis. J Rheumatol 19 (6):968973 Blaszczyk M, Jablonska S (1999) Linear scleroderma en coup de sabre. J Rheumatol 30 (9):19972004 Blaszczyk M, Krysicka-Janiger K, Jablonska S (2000) Primary atrophic profound linear sclero- derma. Ann Dermatol Venereol 126 (10):691694 Boelsma E, Pavel S, Ponec M (1995) Efects of calcitriol on fbroblasts derived from skin of sclero- derma patients. The Journal of der- 4 matological treatment 20 (1):1013 Canizares O, Sachs P, Jaimovich L, Torres V (1958) Idiopathic atrophoderma of pasini and pierini. Dermatology 189 (3):304307 Christianson H, Dorsey C, OLeary P, Kierland R (1956) Localized scleroderma: A clinical study of twohundred thirty-fve cases. Arch Dermatol 74:629639 Chung H, Hann S (1997) Lupus panniculitis treated by a combination therapy of hydroxycvhloro- quine and quinacrine. J Am Acad Dermatol 30 (6):937943 David J, Wilson J, Woo P (1991) Scleroderma En coup de sabre. Medicine (Baltimore) 73 (5):241245 Diaz-Perez J, Connolly S, Winkelmann R (1980) Disabling pansclerotic morphea of children. Med Clin North Am 73 (5):11571166 Drago F, Rampini P, Lugani C, Rebora A (1998) Generalized morphoea afer antitetanus vaccina- tion [letter]. Arch Dermatol 121 (3):345347 Fleischmajer R, Nedwich A (1972) Generalized morphea. J Invest Dermatol 58 (3):129132 Fleischmajer R, Prunieras M (1972) Generalized morphea. J Invest Dermatol 76 (5):400403 Foeldvari I (1998) Progressive linear scleroderma and morphea in a child. J Rheumatol 19:956958 Fujimoto M, Sato S, Ihn H, Takehara K (1995) Autoantibodies to the heat-shock protein hsp73 in localized scleroderma. J Eur Acad Dermatol Venereol 10 (3):283284 Garzon M, Paller A (1999) Ultrapotent topical corticosteroids treatment of childhood genital li- chen sclerosus. Association with type i procollagen and infammatory cell markers in difuse and limited systemic sclerosis, morphea, and raynauds phenomenon. Dermatology 199 (2):185186 Jablonska S (1975a) Facial hemiatrophy and its relation to localized scleroderma. Polish Medical Publishers, Warsaw, pp 277303 Jablonska S, Rodnan G (1979) Localized forms of scleroderma. Clin Rheumatol Dis 5:215241 Jablonska S, Blaszczyk M (2004) Is superfcial morphea synonymous with atrophoderma pasini- pierini? J Immunol 183 (9):54585467 Jessop S, Whitelaw D (2009) Methotrexate for sclerotic skin disorders: No evidence for efective- ness. Br J Dermatol 161 (5):1205 Joly P, Bamberger N, Crickx B, Belaich S (1994) Treatment of severe forms of localized sclero- derma with oral corticosteroids: Follow-up study on 17 patients. Acta Derm Venereol 80 (1):2627 Kencka D, Blaszczyk M, Jablonska S (1995) Atrophoderma pasini-pierini is a primary atrophic abortive morphea. Dutz Kerscher M, Dirschka T, Volkenandt M (1995a) Treatment of localised scleroderma by uva1 pho- totherapy. Pediatr Dermatol 8 (4):292295 Kobayasi T, Serup J (1985) Vascular changes in morphea. Acta Derm Venereol 65 (2):116120 Kobayasi T, Willeberg A, Serup J, Ullman S (1990) Generalized morphea with blisters. Arch Dermatol 145 (9):10171022 Kubo M, Ihn H, Yamane K, Tamaki K (2001) Up-regulated expression of transforming growth factor beta receptors in dermal fbroblasts in skin sections from patients with localized sclero- derma. Pediatr Dermatol 14 (2):113116 Micalizzi C, Parodi A, Rebora A (1994) Morphea with nodular lesions. Br J Dermatol 131:298300 Micalizzi C, Parodi A, Rebora A (1996) Generalized bullous morphoea. American Journal of Diseases of Children (1960) 146 (6):733736 Mitchet C, Doyle J, Ginsburg W (1981) Eosinophilic fascitis: Report of 15 cases. J Dermatol 26 (1):1117 Mohrenschlager M, Jung C, Ring J, Abeck D (1999) Efect of penicillin g on corium thickness in linear morphea of childhood: An analysis using ultrasound technique. Pediatr Dermatol 16 (4):314316 Moller R, Serup J, Ammitzboll T (1985) Glycosaminoglycans in localized scleroderma (morphoea). Proc R Soc Med 66 (11):10831085 Nagai M, Hasegawa M, Takehara K, Sato S (2004) Novel autoantibody to cu/zn superoxide dis- mutase in patients with localized scleroderma. J Invest Dermatol 122 (3):594601 Narvaez D, Kanitakis J, Faure M, Claudy A (1996) Immunohistochemical study of cd34-positive dendritic cells of human dermis. Cutis 22 (5):569572 Neuhofer J, Fritsch P (1984) Treatment of localized scleroderma and lichen sclerosus with etreti- nate. Arch Dermatol 145 (2):127130 OLeary P, Montgomery H, Ragsdale W (1957) Dermatohistopathology of various types of sclero- derma. Arch Dermatol 75:7887 Oikarinen A, Knuutinen A (2001) Ultraviolet a sunbed used for the treatment of scleroderma. J Rheumatol 24 (1):7380 Rai R, Handa S, Gupta S, Kumar B (2000) Bilateral en coup de sabre-a rare entity.

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