IL-6 blockade reverses the abnormal STAT activation of peripheral blood leukocytes from rheumatoid arthritis patients.

Considering the interplay of multiple STATs in response to cytokines, we investigated how IL-6 and its blocking affect STAT signaling in rheumatoid arthritis (RA). Leukocytes obtained from RA patients before and after tocilizumab treatment and healthy donors (HDs) were cytokine-stimulated and STAT phosphorylation was analyzed by cytometry. RA patients had significantly fewer pSTAT1+, pSTAT3+, and pSTAT6+ monocytes and pSTAT5+ lymphocytes than HDs. After 24weeks of treatment, percentages of IFNγ-induced pSTAT1+ and IL-10-induced pSTAT3+ monocytes in RA patients increased, reaching levels comparable to HDs. pSTAT1+ and pSTAT3+ cells correlated inversely with RA disease activity index and levels of pSTAT+ cells at baseline were higher in patients with good EULAR response to tocilizumab. IFNγ-induced pSTAT1+ cells correlated inversely with memory T cells and anti-CCP levels. IL-10-induced pSTAT3+ cells correlated with Treg/Teff ratio. Our findings suggest that IL-6 blocking reduces the inflammatory mechanisms through the correction of STAT1 and STAT3 activation status.

Effectiveness of ustekinumab in patients with psoriatic arthritis in a real-world, multicenter study.

OBJECTIVE: To assess the effectiveness and survival of ustekinumab (UST) among patients with psoriatic arthritis (PsA) treated under routine clinical care. METHODS: Multicenter study. Epidemiological and clinical data was collected through electronic medical records of all patients with PsA who started UST in 15 hospitals of Spain. RESULTS: Two hundred and one patients were included, 130 (64.7%) with 45 mg and 71 (35.3%) with 90 mg. One hundred and thirty one patients (65.2%) had previously received another biological therapy. The median baseline DAS 28 ESR was 3.99, and Psoriasis Area and Severity Index (PASI) was 3. Overall, there was a significant decrease in DAS66/68 CRP, swollen joint count (SJC), tender joint count (TJC), and PASI in the first month of treatment, with earlier improvement in skin (PASI) than joints outcomes. Survival was numerically lower in patients with UST 45 mg (58.1%) than 90 mg (76.1%), although significant differences were not found (p = 0.147). When comparing naïve and < 1 TNF blocker versus > 2 TNF blocker-experienced patients, a significantly earlier response was seen in the former group regarding SJC (p = 0.029) at 1 month. Fifty-one patients (25.3%) stopped UST due to joint inefficacy and 4 patients due to adverse events (1.9%). Drug survival was significantly better in patients with fewer lines of previous biological agents (p = 0.003 for < 1 TNF blocker versus > 2 TNF blocker users). CONCLUSIONS: UST was effective in PsA patients in a routine clinical care setting. Patients with UST 90 mg and fewer lines of previous biologics achieved better and faster responses. Key Points • Largest cohort of patients with PsA in treatment with UST with specific rheumatological indication. • First cohort of patients with PsA comparing effectiveness of UST according to 45/90 mg dose.

Concurrent infection with Trichinella spiralis and other helminths in pigs.

The aim of this study was to investigate possible influence of different helmintosis in the development of Trichinella spiralis in experimental infected pigs. Forty-two Iberian pigs were allocated to six groups. Three groups were single inoculated with Ascaris suum, Metastrongylus apri or T. spiralis, respectively. Two groups were co-infected with T. spiralis and A. suum or T. spiralis and M. apri, respectively, while the last group included uninfected control pigs. Clinical signs were only observed in pigs with single or concurrent M. apri infections, with more severe respiratory symptoms in pigs with mixed M. apri infection. The number of A. suum and M. apri lung larvae, intestinal larvae of A. suum and adult M. apri were reduced in pigs with mixed Trichinella infections compared to pigs with single infections. In contrast, the number of liver white spots was higher in pigs with mixed infections. While T. spiralis muscular larval burdens were increased in pigs concomitantly infected with M. apri, they were reduced in pigs concomitantly infected with A. suum, compared to pigs receiving single infections with either of these helminths. Pigs with single or mixed A. suum infections showed higher eosinophil levels compared to the remaining groups. IgGt, IgG1, IgG2 and IgM against T. spiralis antigen could not be detected in pigs with single Ascaris or Metastrongylus infections, indicating that no cross-antibodies were produced. IgGt, IgG1 and IgM antibodies were detected earlier and generally at higher levels in mixed T. spiralis infections compared to single T. spiralis infections. The results suggest that T. spiralis had a low synergistic interaction with M. apri in concomitantly infected pigs, and an antagonistic interaction in concurrent infection with A. suum.

Expression of phenotypical changes by human breast epithelial cells treated with carcinogens in vitro.

In order to determine whether human breast epithelial cells undergo malignant transformation when treated with chemical carcinogens in vitro, and how host factors, such as degree of gland development, affect their response to that treatment, twenty-two reduction mammoplasty specimens from women ranging in age from 18 to 63 years and with different parity history were studied in their morphology and in their response to carcinogen treatment in vitro. Fixed tissues were processed for whole mount preparations; it was found that the lobules of the nonpregnant or resting breast tissue could be classified into three types based upon their size and number of alveolar buds composing each one of them, lobules type 1 being the least and lobules type 3 the most differentiated ones. Breast tissues were classified according to the relative proportion of lobules composing it and the reproductive and clinical history of the patient into groups A, B, or C. Fresh tissues from these three groups were digested for obtention of mammary epithelium organoids, which were plated in Dulbecco's minimal essential medium:Ham's F12 (1:1), with 5% horse serum to enhance attachment. When they reached confluence, the cells were replated in serum-free medium; 48 h postplating they were treated with 1.0 microgram/ml 7,12-dimethylbenz(a)anthracene for 24 h, or with 1.0 microgram/ml N-methyl-nitrosourea for 3 h. At every passage, the cells were monitored for colony formation efficiency, survival efficiency in agar-methocel, multinucleation assay, karyotyping, immunocytochemical detection of tumor associated antigens, lectin reactivity, and tumorigenic assay by injection into nude mice. By the fourth passage after treatment, both carcinogens induced increased survival, colony formation in agar-methocel, multinucleation, and increased reactivity with the tumor associated antigen B1:1, an epitope of carcinoembryonic antigen, and the lectins concanavalin A, Dolichos biflorus, and soybean agglutinin. This response was observed only in human breast epithelial cells obtained from less differentiated breasts (Groups A and B), whereas human breast epithelial cells from breasts exhibiting good lobular development (Group C) did not exhibit those changes. No karyotypic abnormalities were detected in treated cells, and they failed to induce tumors when injected into nude mice. It was concluded that mammary gland development is independent of age but is strongly influenced by both the reproductive and clinical history of the donor.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of Zoonotic Protozoa Toxoplasma gondii and Sarcocystis suihominis in Wild Boars from Spain.

Food safety regulations require the control of the presence of protozoa in meats destined for human consumption. Wild boar (Sus scrofa) meat may constitute a source of zoonoses. A 23.8% (688/2881) seroprevalence of anti-Toxoplasma gondii antibodies and 72.2% (662/910) Sarcocystis sarcocysts prevalence were detected among wild boars hunted in Southwestern areas of Spain. Identity of Sarcocystis spp. was performed by RFLP-PCR and sequencing, detecting S. miescheriana (7/8) and the zoonotic S. suihominis (1/8). Risk assessment studies of these coccidian in meats destined to human consumption are needed.

Detection of Leishmania DNA and blood meal sources in phlebotomine sand flies (Diptera: Psychodidae) in western of Spain: Update on distribution and risk factors associated.

Leishmaniosis caused by Leishmania infantum is present in Mediterranean countries, with high prevalence in areas of the center and south of Spain. However, in some regions such as Extremadura (in southwest of Spain), data has not been updated since 1997. The aim of this work was (i) to provide information about the distribution of phlebotomine sand fly species in western of Spain (Extremadura region), (ii) to determine risk factors for the presence of sand fly vectors and (iii) to detect Leishmania DNA and identify blood meal sources in wild caught females. During 2012-2013, sand flies were surveyed using CDC miniature light-traps in 13 of 20 counties in Extremadura. Specimens were identified morphologically and females were used for molecular detection of Leishmania DNA by kDNA, ITS-1 and cyt-B. In addition, blood meals origins were analyzed by a PCR based in vertebrate cyt b gene. A total of 1083 sand flies of both gender were captured and identified. Five species were collected, Phlebotomus perniciosus (60.76%), Sergentomyia minuta (29.92%), P. ariasi (7.11%), P. papatasi (1.48%) and P. sergenti (0.74%). The last three species constitute the first report in Badajoz, the most southern province of Extremadura region. Leishmania DNA was detected in three out of 435 females (one P. pernicious and two S. minuta). Characterization of obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae in S. minuta and L. infantum in P. perniciosus. Haematic preferences showed a wide range of hosts, namely: swine, humans, sheep, rabbits, horses, donkeys and turkeys. The simultaneous presence of P. perniciosus and P. ariasi vectors, the analysis of blood meals, together with the detection of L. infantum and in S. minuta of L. tarentolae, confirms the ideal conditions for the transmission of this parasitosis in the western of Spain. These results improve the epidemiological knowledge of leishmaniosis and its vectors in this part of Spain, highlighting the need for ongoing entomological and parasitological surveillance.

Epidemiology of Oestrus ovis (Linneo, 1761) infestation in goats in Spain.

This survey was conducted to determine the chronobiology and seroprevalence of nasal bot infestation (Oestrus ovis) in Spain and to identify the risk factors associated with this disease in caprine herds. A total of 1590 sera from adult goats were collected at random on 175 farms in southwestern Spain. Sera were tested by ELISA, using crude protein from second stage larvae as antigen. The mean seroprevalence was 46.04% and mean percentage of optical densities was 41.83. These data indicate a high prevalence of this parasite in the investigated areas. The serological survey revealed that goats managed at higher altitudes, at meridians latitudes and on farms with small herds had a smaller probability of infestation. Eighty goat heads, obtained from abattoirs in the central region of Spain, were collected and examined for nasal botflies from February to October 2002. O. ovis larval stages were recovered from the nasal-sinus cavities of 23 goats, reaching a prevalence of 34.94%. The mean larval burden was 3.9 larvae per infested head. No first instars were found during February and March, when the second instar reached its larger count. The third instar was observed in very small number during the whole period of study, with one peak occurring in July--August. These data show the existence of a favourable period for the development of larval instars of O. ovis in goats that starts in February and finishes in September.

Evidence of interaction between Ascaris suum and Metastrongylus apri in experimentally infected pigs.

A study has been carried out with the aim to determine possible interactions between Ascaris suum and Metastrongylus apri under experimentally infected pigs. Twenty-eight Iberian pigs were allocated into four groups. Group 1 was inoculated with 5000 infective A. suum eggs; group 2 received concurrently 5000 infective A. suum eggs and 5000 infective M. apri larvae; group 3 received 5000 infective M. apri larvae; group 4 served as uninfected controls. In each group, pigs were necropsied on day 7 (n = 4) and day 28 (n = 3) post-infection (p.i.). Pigs with single M. apri infections showed earlier and more severe respiratory symptoms compared to pigs with mixed infection, while no clinical signs were observed in pigs single infected with A. suum. Mean burdens of immature A. suum and immature and adult M. apri were reduced in pigs with concomitant infection both on day 7 and 28 p.i., respectively. In contrast, the number of white spots was significantly increased on day 7 in pigs with mixed infection. In addition, pigs of group 1 showed the highest eosinophil levels in blood compared to pigs in groups 2 (intermediate levels) and 3 (moderate levels). The results suggest an antagonistic interaction between A. suum and M. apri in concomitantly infected pigs.

Method for collecting Hypoderma spp. (Diptera: Oestridae) larvae from cattle.

A new method for collecting 3rd-instar Hypoderma lineatum (Villers) and Hypoderma bovis (L.) is described. A procedure for carrying out chronobiological and other studies involving the life cycle of these flies is included. The larvae are collected from cattle using a dressing made of a circle of cardboard attached to a piece of tulle similar in color to the coat of the animal. These small cloth cages were attached to the animal skin with fast-drying contact glue wherever the swellings produced by 2nd- or 3rd-instar Hypoderma spp. had been previously detected. Each animal was inspected daily to detect the appearance of new larvae. This procedure allows the collection of each emerged larva without contact with other larvae and avoids friction of the cloth cages or dressing with the larvae, thus reducing larval death and increasing viability of the parasite.

Experimental bovine hypodermosis in Spain.

An experimental survey was carried out in western Spain to investigate both the chronobiology of Hypoderma spp. and the immunoresponse of their bovine hosts. This study was initiated with a new system of obtaining Hypoderma spp. larvae directly from their host, including the eclosion of adults from their pupae, infestation under natural but controlled conditions, and confirmation of the resulting infection. This survey was carried out over 2 cattle grub seasons; it was possible to infest and reinfest the experimental animals and to monitor them by both parasitological methods and by an enzyme-linked immunosorbent assay. This method permitted the evaluation of the development of anti-Hypoderma antibodies during the experiment. The experimental design also enabled us to establish the period of detectable H. lineatum infection to be from December until the end of April with the largest number of warbles observed during March and April. After a pupal period of < 30 d, adults were seen in April and May. Hypoderma bovis (de Geer) showed a delay of 2 m.o. relative to H. lineatum (de Villiers). This study reports a completed biological life cycle of Hypoderma spp. under controlled conditions in both natural and experimental environments.

Infant nutrition.

Although most physicians agree that human milk is superior to artificial feeding, the latter is far more popular in well developed countries. There are various reasons for the decrease in breast feeding but a major factor has been the introduction of a variety of simple, safe milk and milk-substitute formulas. The healthy full-term infant will thrive on any of a number of properly prepared formulas as well as human milk. Mothers should be encouraged to breast feed by the obstetrician and pediatrician, but should not be made to feel guilty if they do not. Regardless of the method used for feeding, proper instruction is mandatory. A practical approach to the management of breast feeding is presented, as is a guide to artificial feeding. A brief review of the full-term infant's nutritional requirements is given together with a comparison of human milk, cow's milk, and commercially prepared feeding products. The availability and use of the specialized formulas is also included.

Antibody response and duration of latent infection in sheep following experimental infection with Babesia ovis.

Babesia ovis isolated in Extremadura (Spain) was the subject of a serological study in experimentally inoculated sheep. The first antibody titres, determined by the indirect fluorescent antibody (IFA) test, were observed 7-8 days post infection (p.i.) in all animals except the splenectomized group, in which the only animal that survived showed antibody response 10 days p.i. A faster response following challenge was observed in sheep which were seropositive before inoculation, which suggests the existence of an antigen memory. The highest titres were reached 16-25 days p.i., and subsequently began to fall, reaching minima at the end of the experimental period (330 days p.i.). The chronic carrier state in experimental B. ovis infection had a duration of at least 2 years. Passive transmission of antibodies from experimentally infected mothers to newborn lambs was also detected. Antibody levels were observed for a period not longer than 2 months after birth.

Isolation and identification of Babesia ovis in Extremadura (Spain).

A morphological and morphometric comparison was made of a Babesia ovis strain isolated in Extremadura (Spain) and a B. ovis strain from Turkey. Strains were morphologically similar, with a clear predominance of single sub-spherical or anaplasmoid forms. The use of an image analyser to measure B. ovis merozoites revealed significant differences in the long axis, short axis and volume between the two strains. Both strains however behaved in a similar manner in the indirect fluorescent antibody (IFA) test, which suggests a high degree of common antigenicity, despite morphometric differences. Conversely, no cross-reaction was observed between these strains of B. ovis and the other antigens used: B. motasi (The Netherlands), B. motasi (Turkey), B. crassa (Iran) and Theileria ovis (Turkey). B. ovis is therefore considered to be the aetiological agent of babesiosis in the area under study.

Histopathological changes in sheep experimentally infected with Babesia ovis.

Histopathological study was made of 12 Merino sheep - five splenectomized and seven intact - experimentally infected with Babesia ovis. Non-purulent encephalitis; initially exudative and subsequently interstitial pneumonia; pericarditis, myocarditis and haemorrhagic endocarditis; centrilobular necrotic hepatitis; hyperplasia of the lymphoreticular system; necrosis and vascular changes in adrenal glands were observed. The kidney was the most severely affected organ, exhibiting acute tubular necrosis typical of kidney shock syndrome. The lesions observed were suggestive of hypovolemic shock culminating in haemorrhagic diathesis owing to consumptive coagulopathy. Additionally, the massive release of catabolites from lysis and necrosis apparently produced endotoxic shock.

Efficacy of doramectin against naturally acquired nematode infection in Iberian swine.

Studies were carried out to determine the therapeutic efficacy of doramectin, administered intramuscularly at a dose of 300 microg/kg live weight, against naturally acquired helminths of extensively farmed Iberian pigs. The first study (slaughter study) evaluated, through necropsy of the study animals, the product's efficacy against gastrointestinal and pulmonary nematodes (Ascaris suum, Oesophagostomum dentatum and Metastrongylus sp.) whilst the second, faecal egg count reduction study, (FECR study) evaluated the drug's efficacy only against gastrointestinal helminths (A. suum, Trichuris suis and Oesophagostomum sp.). The first study used 20 animals divided into two equal groups of 10 on the basis of body weight and faecal egg count. One group constituted saline treated controls and the other was doramectin treated. On Day 14 post treatment half of the animals in each group were necropsied and the number of parasites present counted. On Day 15 the remaining half of each group underwent the same procedure. The second study was carried out with 40 animals divided equally into two groups of 20. This study determined the effect of doramectin treatment on faecal egg counts as an indicator of parasite burden. The first study demonstrated an efficacy of 100% against adult Metastrongylus sp. and A. suum, whilst the efficacy against O.dentatum was 96.3%. The second study indicated that at Day 21 post treatment there was a 100% reduction in egg counts in faeces in comparison to untreated controls.

Hypoglycaemia in experimental canine trichinellosis.

This paper studies changes in serum glucose levels in dogs experimentally infected with Trichinella spiralis, and the histological causes of those changes. Hypoglycaemia is evident in 100% of experimental animals, and can be attributable to three factors: the reduced absorptive capacity of the intestine due to lesions (catarrhal and desquamative enteritis), high glucose consumption by parasites, or histopathological changes in the liver (glycogenic degeneration).

Seasonal variations in the larval burden distribution of Oestrus ovis in sheep in the southwest of Spain.

A chronobiological study of oestrosis was conducted for larval instars of Oestrus ovis from November 2000 to September 2002 with the examination of 477 adult sheep of the southwest region of Spain. Skulls from slaughtered sheep were examined and the different O. ovis larval stages (L1, L2, L3) were recovered from the nasal-sinus cavities. O. ovis larvae were detected in 339 sheep, reaching a prevalence of 71.1%. Only one farm was free of infested sheep indicating a prevalence of the 97.91% among studied flocks. The mean larval burden was 18.54 larvae per infested head during the coldest months in the southwest of Spain when the larval burden reached its highest levels, especially of the first larval stage (L1). However, the maximum percentage of L1 coincided with the minimum percentage of the second larval stage (L2). The third larval stage (L3) was observed in relatively low levels during the entered study period, but two peaks occurred in April-May and in September-October. During the 2 years of sampling, all the different larval stages were simultaneously recovered throughout the year, indicating the existence of a long favourable period for the evolution and development of the larval instars, which would start between February and March and finishing in November.

Presence of immunoglobulins and antigens in serum, lung and small intestine in Ascaris suum infected and immunised pigs.

The immunodetection of local Ascaris suum antigens and local and systemic antibodies were analysed in pigs reinfected with eggs or immunized with the 14, 42 and 97 kilodalton (kDa) fractions from A. suum. Twenty-one Iberian pigs were divided in 7 groups of 3 pigs. Groups 1 and 2 were uninfected and challenge control groups, respectively. Groups 3 and 4 were infected weekly with increasing doses of A. suum eggs and Group 4 was additionally treated with pyrantel pamoate. Groups 5, 6 and 7 were immunised with the 14, 42 or 97 kDa fractions from adult worms, respectively. Groups 2-7 were challenged with 10,000 infective eggs. Animals of Groups 3 and 4 showed a pulmonary granulomatous reaction with moderate number of eosinophils and leukocytes, while Groups 5-7 presented higher number of cells, especially in animals immunized with the 42 kDa fraction. These immunized groups presented abundant deposition of Ascaris body fluid (BF) and body wall (BW) antigens as well as the 14 and 42 kDa fractions in the pulmonary and intestinal tissues, while lower deposition of antigens was observed in animals of Groups 3 and 4. The immunized pigs of Groups 5 and 6 showed the highest systemic IgG titres in serum and these antibodies were negatively correlated with the number of larvae recovered in the lungs, suggesting that the IgG response may have a protective function against the ascariosis. The highest concentrations of IgA-bearing cells were observed in animals of Groups 3 and 4 compared to the immunised pigs (Groups 5-7), suggesting that local IgA production may be involved in the protection against migrating larvae. The main localisations of IgA-bearing cells were the bronchial and peribronchial areas of lungs and the lamina propia of duodenum. Low numbers of local IgG-bearing cells were observed in all animals and no IgM-bearing cells were detected in the local tissues.

Experimental trichinellosis in goats.

The susceptibility and distribution of Trichinella spiralis infection in goats were examined in ten autochthonous kids, 2 months old and about 10 kg body weight. The animals were divided into two groups: one experimental group with eight animals, infected with 10,000 T. spiralis 'T1' encysted larvae and a control group with two non-infected animals. All the animals of the experimental group infected by the parasite showed that Trichinella larvae have a special affinity for the tongue, masseters, diaphragm, flexor-extensor muscles, intercostal muscles and myocardium in decreasing order. The ELISA test carried out showed the first increments of optical density (OD) on Day 16 postinfection (p.i), peaking on Days 37-44 p.i. and remaining elevated from this day on, with a slight fall at the end of the experiment (Day 90 p.i.). No alterations were observed in the OD obtained in control animals throughout the experiment. The great muscular establishment of T. spiralis larvae and the sigmoidal evolution of antibody levels confirm the host character of the goat to the parasite.