PSC833, cyclosporine analogue, downregulates MDR1 expression by activating JNK/c-Jun/AP-1 and suppressing NF-kappaB.

PURPOSE: Multidrug resistance (MDR) is one of the major causes of clinical cancer chemotherapy failure. PSC833 is well known as a non-immunosuppressant cyclosporine analogue that functionally inhibits P-glycoprotein (Pgp), a product of the MDR1 gene. We investigated whether PSC833 could also alter MDR1 expression and, if so, which mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways were involved in this event. METHODS: MTT assay and flow cytometry were used for the analysis of cytotoxicity and intracellular drug accumulation, respectively. RT-PCR and Western blot assays for analysis of gene expression and electrophoretic mobility shift assays for determination of DNA-binding activity of transcription factors were used. RESULTS: The doxorubicin-resistant lung cancer cell subline (SK-MES-1/DX1000), selected from SK-MES-1/WT cells, upregulated MDR1 expression, thereby showing MDR phenotypes. PSC833 sensitized SK-MES-1/DX1000 cells to doxorubicin. PSC833 (5 microM) also decreased the intracellular accumulation of fluorescent Pgp substrates such as rhodamine 123 and daunorubicin in SK-MES-1/DX1000 cells. PSC833 downregulated MDR1 mRNA and Pgp expression in a time- and concentration-dependent manner. PSC833 activated c-Jun NH2-terminal kinase (JNK)/c-Jun and enhanced AP-1 DNA-binding activity, but suppressed nuclear translocation of NF-kappaB, all of which were prevented by pretreatment with a JNK inhibitor SP600125. CONCLUSIONS: These results indicate that PSC833 not only sensitizes SK-MES-1/DX1000 cells to doxorubicin by enhancing drug accumulation, but also downregulates MDR1 expression by activating JNK/c-Jun/AP-1 and suppressing NF-kappaB.

Quercetin inhibits expression of inflammatory cytokines through attenuation of NF-kappaB and p38 MAPK in HMC-1 human mast cell line.

OBJECTIVE AND DESIGN: Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce production of pro-inflammatory cytokines with immune regulatory properties. We investigated the effect of quercetin on the expression of pro-inflammatory cytokines in human mast cell line, HMC-1. METHODS: HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 (PMACI). RESULTS: Quercetin decreased the gene expression and production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and IL-8 in PMACI-stimulated HMC-1 cells. Quercetin attenuated PMACI-induced activation of NF-kappaB and p38 mitogen-activated protein kinase. CONCLUSION: Our study provides evidence that quercetin may suitable for the treatment of mast cell-derived allergic inflammatory diseases.

Spirocerca lupi in dogs: prophylactic effect of doramectin.

Spirocerca lupi is primarily a parasite of dogs, which typically causes oesophageal nodules, aortic aneurysms, and spondylitis. This study investigated the efficacy of doramectin as a prophylactic agent for canine spirocercosis. Five beagle dogs were injected subcutaneously with doramectin (400 microg/kg on 3 occasions 30 days apart q30d), while 5 other beagle dogs served as untreated controls. All dogs were inoculated with 40 infectious S. lupi larvae (L3) one month after the last doramectin treatment. All control dogs and 4/5 treated dogs became infected. Two control dogs died of ruptured aortic aneurysms, while no deaths occurred in treated dogs. Oesophageal nodules appeared 40-103 day later in treated as compared to control dogs, and eggs appeared in the faeces 49-106 day later in treated as compared to control dogs. The mean faecal egg count on day 223 in the treatment group was reduced by 99.77%. All control dogs had thoracic radiographic changes during the study, while only 2/5 study dogs showed radiographic changes. This study shows that although doramectin did not entirely prevent canine spirocercosis it reduced the clinical signs associated with infection and delayed and reduced egg output.

Retrospective study of 46 cases of feline haemobartonellosis in Israel and their relationships with FeLV and FIV infections.

Forty-six cats with clinical haemobartonellosis were studied; 75 per cent of the cats of known age were two-and-a-half years old or younger, 50 per cent were intact males and 19.5 per cent were castrated males. The predominant signs of the disease were tachypnoea, lethargy, depression, anorexia, infestation with fleas, pale mucous membranes, icterus, emaciation, dehydration, splenomegaly, anaemia, leucocytosis, increased activities of alanine aminotransferase and aspartate aminotransferase, and azotaemia. Thirty-eight per cent of the cats that were tested for feline leukaemia virus (FeLV) antigen were positive, and 22 per cent of those tested for feline immunodeficiency virus (FIV) antibodies were positive. The prevalence of both FeLV and FIV was much higher than in the general Israeli cat population. The cats infected with both Haemobartonella felis and FeLV had a significantly lower body temperature, were more anaemic and the mean cell volume of their erythrocytes was greater than in the cats with haemobartonellosis alone.

Development of hypertrophic osteodystrophy and antibody response in a litter of vaccinated Weimaraner puppies.

Two different vaccination protocols were compared with regard to the development of hypertrophic osteodystrophy (HOD) (also termed metaphyseal osteopathy) and effectiveness of immunisation in a litter of 10 Weimaraner puppies. Five puppies (group 1) were vaccinated with a modified live canine parvovirus vaccine (CPV) and then two weeks later with a trivalent vaccine containing modified live canine distemper virus and adenovirus type 2 combined with a Leptospira bacterin (DHL). The CPV and DHL vaccine protocols were administered a further two times, at two-week intervals. Group 2 was vaccinated with three consecutive multivalent vaccines containing modified live canine distemper virus, canine parvovirus, parainfluenza and adenovirus type 2 combined with a Leptospira bacterin, at four-week intervals. All puppies were first vaccinated at the age of eight weeks. Three dogs in group 1 developed HOD, while all five dogs in group 2 developed HOD during the study period. Dogs in group 2 had more episodes of HOD than those in group 1. Dogs in group 1 developed higher antibody titres to canine distemper virus and parvovirus compared with those in group 2. Only two out of the 10 dogs developed protective antibody titres to parvovirus. The results of this study suggest that the two different vaccination protocols affected the pattern of appearance of HOD and immunisation in this litter of Weimaraner puppies. The results obtained and the previously reported data suggest that a larger controlled study is needed to further elucidate the effect of different vaccination protocols on HOD and immunisation in Weimaraner puppies.

Presence of immune-complexes, and absence of antinuclear antibodies, in sera of dogs naturally and experimentally infected with Ehrlichia canis.

Antinuclear antibodies (ANA), immunoglobulin G (IgG) concentrations and circulating immune-complexes (CIC) were measured, over a period of 3 years, in 6 dogs experimentally infected with Ehrlichia canis, and in 10 dogs naturally infected with the rickettsia. No ANA were detected in any of the samples tested. The IgG concentrations were shown to be higher in the infected dogs when compared to the control dogs. CIC were detected in 2 of 10 naturally and 2 of 6 experimentally infected dogs, during both the acute and the subclinical phases of the disease. The results of this study suggest that ANA do not play a role in the pathogenesis of CME. It is however suggested that some manifestations in canine ehrlichiosis are immune-complex mediated.

Dynamics of IgG1 and IgG2 subclass response in dogs naturally and experimentally infected with Ehrlichia canis.

Immunoglobulin (Ig) G subclasses were measured in dogs naturally and experimentally infected with Ehrlichia canis using enzyme-linked immunosorbant assay (ELISA). In this study, a higher IgG2 subclass response was noticed to natural and experimental E. canis infection in dogs. Anti-E. canis-IgG2 optic density (OD) values were found to be significantly higher than anti-E. canis-IgG1 during the different phases of the disease, and no differences in the IgG subclass responses to E. canis infection were found between symptomatic and asymptomatic dogs. Doxycycline treatment, which eliminated the rickettsia in three of four persistently infected dogs, had no noticeable influence on the E. canis-IgG subclass OD values during the treatment period. In order to facilitate the study, an ELISA for the detection of anti-E. canis IgG was developed and was shown to be sensitive and specific for E. canis-IgG, and in a significant correlation with the indirect immunofluorescence antibody test.

Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis.

Dogs are susceptible to a number of ehrlichial diseases. Among them, canine monocytic ehrlichiosis is an important and potentially fatal disease of dogs caused by the rickettsia Ehrlichia canis. Diagnosis of the disease relies heavily on the detection of antibodies and is usually carried out using the indirect immunofluoresence antibody (IFA) test. The IFA test may be confounded by cross-reactivities between a number of the canine ehrlichial pathogens. This article presents a review of the ehrlichial diseases affecting dogs with reference to their immune responses, host specificities, cross-reactivites and diagnosis. Diagnostic means such as Western immunblot, dot-blot and PCR are discussed. The use of the IFA test as a diagnostic means for E. canis is presented along with its potential pitfalls. The review emphasizes that the disease process, cross-reactivites with other ehrlichial species, multiple tick-borne infections and persistent IFA antibody titers post-treatment, should all be considered when interpreting E. canis serological results.

Detection of platelet-bound antibodies in beagle dogs after artificial infection with Ehrlichia canis.

Six dogs were infected with Ehrlichia canis by intravenous injection of heavily infected DH82 cells. All dogs developed typical signs of canine monocytic ehrlichiosis. Using flow cytometric technology, platelet-bound IgG (PBIgG) were detected in 5 of the 6 dogs after experimental infection with E. canis over a period of 3-10 days post infection (PI). The first detection of PBIgG was made as early as day 3 PI in 2 out of 6 dogs, and on day 5 PI in 1 dog. On day 7 PI, PBIgG was detected in 2 dogs, and on day 10 PI in 3 out of 6 dogs. This is the first report documenting the presence of PBIgG following E. canis infection in dogs. This finding further supports the theory that the thrombocytopenia seen in canine monocytic ehrlichiosis has an immunological component and that exposure to an infectious agent, in this case the rickettsia E. canis, can trigger autoimmune mechanisms. Due to the heterogeneous appearance of PBIgG among the infected dogs it was concluded that other non-immunological mechanisms are probably also involved in the pathogenesis of the thrombocytopenia seen in canine monocytic ehrlichiosis.

Therapeutic effect of doxycycline in experimental subclinical canine monocytic ehrlichiosis: evaluation of a 6-week course.

The efficacy of doxycycline treatment (10 mg/kg of body weight every 24 h for 42 days) in eliminating Ehrlichia canis from four subclinically infected dogs was evaluated. One dog remained PCR positive, suggesting that 6 weeks of doxycycline treatment may not be sufficient to clear E. canis parasites from all subclinically infected dogs. Serology (indirect immunofluorescent antibody assay) was shown to be unreliable in assessing recovery from the carrier state, as anti-E. canis antibodies persisted after elimination of the parasite. Our findings suggest that an increase in the platelet count may be an important indicator for dogs that recover from subclinical ehrlichiosis.

Investigation of splenic functions in canine monocytic ehrlichiosis.

In order to determine the role of the spleen in the pathogenesis of canine monocytic ehrlichiosis (CME), the effect of splenectomy on the course of the acute phase of experimental was investigated. Intact and splenectomized dogs, sero-negative for Ehrlichia canis antibodies, were infected with the Israeli strain of E. canis. Serology, clinical signs and haematological parameters were recorded prior to infection, and over a period of 60 days post infection, and were compared between the intact and the splenectomized dogs. All dogs seroconverted for IFA E. canis antibodies by days 10 to 17 post infection. There did not appear to be any difference in the day of appearance or in the titer of anti-E. canis IgG antibodies, between the splenectomized and intact groups throughout the course of the study. During the acute stage, food consumption (percentage change) was significantly lower in the intact group compared to the splenectomized group (-66.3% and -25.3%, respectively, p < 0.0001). During this period, significant higher body temperatures were measured in the intact group (average of 39.76 degrees C vs. 38.96 degrees C, p < 0.0001). The haematocrit, red blood cell counts, haemoglobin concentrations and platelet counts were significantly lower (p < 0.05) in the intact group when compared to the splenectomized group during the whole course of the study. The clinical and the haematological findings in our study suggest that the disease process was milder in the splenectomized dogs compared to the intact dogs. The results of this study suggest that the spleen plays an important role in the pathogenesis of CME. Splenic inflammatory mediators and/or other splenic substances, are proposed to play a key role in the pathogenesis of the disease. Our results further substantiate the involvement of immune mechanisms in the pathogenesis of CME.

Amplification of ehrlichial DNA from dogs 34 months after infection with Ehrlichia canis.

In order to determine whether dogs in the subclinical phase of canine monocytic ehrlichiosis (CME) are carriers of Ehrlichia canis and to determine the significance of persistent indirect immunofluorescent anti-E. canis antibody titers during this phase, PCR was performed with blood, bone marrow, and splenic aspirates collected 34 months postinoculation from six clinically healthy beagle dogs experimentally infected with E. canis. At least one of the three samples (spleen, bone marrow, and blood) from four of the six dogs was PCR positive. The spleens of all four of these dogs were PCR positive, and the bone marrow and blood of two of the four dogs were PCR positive. Indirect immunofluorescent-antibody titers increased progressively during the first 5 months postinfection, remained high for an additional period of more than 11 months, and declined thereafter, suggesting that the dogs were recovering from the disease. Five of the dogs remained seropositive 34 months postinfection. The data obtained in this study demonstrate for the first time that clinically healthy dogs in the subclinical phase of CME are carriers of the rickettsia. It was shown that dogs can harbor E. canis for years without developing the chronic clinical disease and that dogs can eliminate the parasite and recover from CME without medical treatment. Our findings suggest that the spleen is the organ most likely to harbor E. canis parasites during the subclinical phase and the last organ to accommodate the parasite before elimination. It was concluded that PCR of DNA extracted from splenic aspirates is a reliable method for determining the carrier state of CME.

Clinical manifestations of infectious canine cyclic thrombocytopenia.

This paper describes five naturally occurring clinical cases of infectious canine cyclic thrombocytopenia that were the first serologically confirmed cases of Ehrlichia platys infection in Israel. In the USA this disease is considered subclinical, but the dogs in this study developed distinct clinical abnormalities. The signs observed by the owners included anorexia, lethargy, depression, weight loss and a mucopurulent nasal discharge. The principal findings on physical examination included lymphadenomegaly, pale mucous membranes, fever and the presence of ticks. The main abnormal haematological and biochemical findings included thrombocytopenia, the presence of giant platelets, low haematocrit, monocytosis and low albumin concentrations. All five dogs were less than two years of age, and four were purebred dogs, suggesting that these two factors may be associated with increased risk to infection and clinical disease. Two of the dogs were seropositive to E canis, a finding which is compatible with other reports, and which confirms that combined infections of E platys and E canis are common; it also suggests that E canis infections may contribute to the pathogenesis of E platys. The distinct clinical manifestation of the disease in these five dogs suggests that there may be a different, more virulent strain of E platys in Israel.

Characterization of the subclinical phase of canine ehrlichiosis in experimentally infected beagle dogs.

Beagle dogs were examined during the subclinical phase of canine ehrlichiosis under controlled conditions. Emphasis was placed on gathering data before artificial inoculation with Ehrlichia canis, and comparing these data with those of the subclinical phase of the disease. In this study all dogs were clinically healthy throughout the 6 month examination period. All subclinically infected dogs had IFA antibody titers to E. canis at a dilution varying from 1:2560 to 1:20480. The most prominent haematological finding was mild thrombocytopenia with a concomitant increase in platelet size, seen in eight of the nine dogs examined. Leukocyte counts were statistically significantly reduced in 78% of the dogs, compared with their preinfection values, with 71% of dogs having significantly reduced absolute neutrophil counts. None of the dogs were either leukopenic nor neutropenic. Six of the nine dogs had increased serum gamma-globulin concentrations. No dogs were overtly anemic, although declines in packed cell volume, haemoglobin concentration and total erythrocyte count were detected in an inconsistent manner among the dogs. It was concluded that, the most reliable parameters for judging possible subclinical ehrlichial infection in beagle dogs was mild thrombocytopenia, together with a persistently high antibody titer to E. canis. Hypergammaglobulinemia would increase the suspicion further. Based on the results presented, routine testing of dogs in E. canis endemic areas is recommended in order to identify and treat dogs in the subclinical phase of the disease.

Functional intestinal hypomotility in association with neuronal damage in a dog.

A young, crossbred dog with a 13-month history of progressively worsening, recurrent episodes of vomiting, anorexia, depression and dehydration was diagnosed as having a functional hypomotility disorder of the small intestines. During hospitalisation, the dog's condition improved only when total parenteral nutrition was administered. When fed orally, the dog developed severe gastric dilatation. Intestinal motility was minimal, but was induced by neostigmine. Post mortem findings included dilatation of the duodenum and progressive narrowing of the small bowel up to the ileum. Histopathology revealed various grades of neuronal degeneration and necrosis of the myenteric plexuses throughout the small intestine. Microscopic changes resembled those reported in dysautonomia, but were limited to small intestinal myenteric plexuses. A localised congenital neuronal defect causing a lack of parasympathetic tone is suspected.

Serum protein alterations in canine ehrlichiosis.

Serum protein electrophoresis was performed in 42 dogs with naturally occurring Ehrlichia canis infection and in 15 clinically healthy dogs (control dogs). The infected dogs were found to have a significant hypoalbuminaemia, hyperglobulinaemia and hypergammaglobulinaemia compared to the control dogs (P < 0.001). A polyclonal gammopathy was found in all but one of the infected dogs which presented a monoclonal gammopathy. alpha-1 globulin was lower while alpha-2 and beta-2 globulin concentrations were significantly higher in the infected dogs (P < 0.0001, P < 0.05 and P < 0.005, respectively). The infected dogs were divided into two subgroups according to haematological parameters, defined as pancytopenic (n = 13) and non-pancytopenic (n = 29). When compared, the pancytopenic group revealed significantly lower concentrations of total protein, total globulin and gammaglobulin (P < 0.01, P < 0.05 and P < 0.005 respectively). The lower concentrations of the gammaglobulins coupled with the pancytopenia suggest that the immune state of the pancytopenic E. canis infected dogs is more compromised, and therefore secondary infections should be expected more frequently in these dogs.

Platelet dysfunction associated with experimental acute canine ehrlichiosis.

To determine whether platelet dysfunction occurs in canine ehrlichiosis, platelet aggregation studies in response to collagen/epinephrine, thrombin and adenosine diphosphate (ADP) were carried out by the indirect method, using sera from six dogs experimentally infected with Ehrlichia canis. Samples of serum taken before infection and four and 20 days after infection were tested by incubation with platelet-rich plasma from a seronegative healthy dog. Platelet aggregation was significantly inhibited in five of six infected dogs in response to at least one of the agonists used. A significant increase in preaggregation lag time was recorded in response to collagen/epinephrine in sera taken 20 days after infection from three of five dogs (P < 0.05). When compared with the preinfection values, a significant increase of 45 per cent in the mean preaggregation lag time was detected (P < 0.05). Maximal relative aggregation responses to ADP decreased significantly in one serum sample taken four days and one taken 20 days after infection (P < 0.01) and there was a significantly lower relative slope for one serum sample 20 days after infection (P < 0.05). Maximal relative aggregation responses to thrombin were significantly decreased together with their relative slopes in serum samples from two of four dogs four days after infection (P < 0.05). The results suggest that platelet dysfunction may occur in the acute stage of canine ehrlichiosis, and may be a contributing factor to the tendency to bleed commonly observed in this disease. Antiplatelet antibodies directed against platelet glycoproteins may play a role in the inhibition of platelet aggregation.

Kinetics of serum antiplatelet antibodies in experimental acute canine ehrlichiosis.

The pattern of appearance of serum antiplatelet antibodies during the acute phase of experimental canine ehrlichiosis (Ehrlichia canis) was investigated in six beagles and correlated with the development of thrombocytopenia. The earliest detection of serum antiplatelet antibodies was made on Day 7 post-inoculation in one dog, on Day 13 in three out of six dogs, and on Day 17 post-inoculation in the remaining two dogs. Thrombocytopenia developed in all infected dogs. The results of this study suggest that antiplatelet antibodies play a role in the destruction of platelets in the acute phase of the disease. It is proposed that E. canis infection in dogs alters the immune system resulting in the overproduction of natural antiplatelet antibodies.