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Topic:Immunology

The equine immune system is a complex network of cells, tissues, and organs that work collaboratively to defend against pathogens and maintain homeostasis. It consists of innate and adaptive components, each with distinct functions and mechanisms. The innate immune system provides the first line of defense through physical barriers, phagocytic cells, and the complement system. The adaptive immune system involves lymphocytes, such as B cells and T cells, which generate specific responses to antigens and provide immunological memory. Research in equine immunology explores the interactions between these components, the impact of genetic and environmental factors on immune function, and the development of vaccines and therapeutics. This page gathers peer-reviewed studies and scholarly articles focusing on the mechanisms, regulation, and clinical applications of the equine immune system in health and disease.
Equine macrophage identification with an antibody (Ki-M6) to human CD68 and a new monoclonal antibody (JB10).
Journal of comparative pathology    February 24, 2000   Volume 122, Issue 2-3 145-154 doi: 10.1053/jcpa.1999.0351
Siedek EM, Honnah-Symns N, Fincham SC, Mayall S, Hamblin AS.Monoclonal antibodies (mAbs) recognizing equine macrophages are scarce. The present study compared the immunocytochemical staining of various equine tissues (lymphoid tissue, lung, liver, small intestine, skin and blood leucocytes) by an antibody, Ki-M6, which detects CD68 in human macrophages and dendritic cells, and by a new anti-equine mAb, JB10, with staining produced by two previously described anti-equine macrophage mAbs, CZ2.2 and CZ3.3. Ki-M6 was shown to identify equine macrophages, which had a distribution different from those identified by CZ2.2 and CZ3.3. JB10 identified equine mac...
Comparison of the phenotypes of Streptococcus zooepidemicus isolated from tonsils of healthy horses and specimens obtained from foals and donkeys with pneumonia.
American journal of veterinary research    February 24, 2000   Volume 61, Issue 2 162-166 doi: 10.2460/ajvr.2000.61.162
Anzai T, Walker JA, Blair MB, Chambers TM, Timoney JF.To determine whether streptococcal pneumonia is caused by strains of Streptococcus zooepidemicus similar to those obtained from the tonsils of healthy horses. Methods: 5 tonsils from healthy horses, 8 tracheal washes and 6 lung specimens from foals with pneumonia, and 5 nasopharyngeal swab specimens from donkeys with acute bronchopneumonia. Methods: Variable M-like protectively immunogenic SzP proteins of 5 isolates of S. zooepidemicus from each tonsil and clinical specimen were compared, using immunoblots. The SzP gene of 13 isolates representative of various SzP immunoblot phenotypes from 1 ...
Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA.
Canadian journal of veterinary research = Revue canadienne de recherche veterinaire    February 19, 2000   Volume 64, Issue 1 38-43 
Cho HJ, Entz SC, Deregt D, Jordan LT, Timoney PJ, McCollum WH.A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highl...
Signalling pathway for histamine activation of non-selective cation channels in equine tracheal myocytes.
The Journal of physiology    February 16, 2000   Volume 523 Pt 1, Issue Pt 1 131-138 doi: 10.1111/j.1469-7793.2000.t01-3-00131.x
Wang YX, Kotlikoff MI.1. The signalling pathway underlying histamine activation of non-selective cation channels was investigated in single equine tracheal myocytes. Application of histamine (100 microM) activated the transient calcium-activated chloride current (ICl(Ca)) and sustained, low amplitude non-selective cation current (ICat). The H1 receptor antagonist pyrilamine (10 microM) blocked activation of ICl(Ca) and ICat. Simultaneous application of histamine (100 microM) and caffeine (8 mM) during H1 receptor blockade activated ICl(Ca), but not ICat. Neither the H2 receptor antagonist cimetidine (20 microM) nor...
Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica.
Toxicon : official journal of the International Society on Toxinology    February 15, 2000   Volume 38, Issue 5 633-644 doi: 10.1016/s0041-0101(99)00177-4
Fernandes I, Lima EX, Takehara HA, Moura-da-Silva AM, Tanjoni I, Gutiérrez JM.Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 000-000. This work compared the specificity, ELISA titers and IgG subclass content of the polyvalent antivenom (anti-Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys) of Instituto Clodomiro Picado (Costa Rica) and the bothropic antivenom (anti-Bothrops jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus) of Instituto Butantan (Brazil). The role of IgG(T) and IgGa subclasses in neutralization of some venom toxic activities and the cross neutraliza...
Development and validation of a monoclonal antibody blocking ELISA for the detection of antibodies against both equine herpesvirus type 1 (EHV1) and equine herpesvirus type 4 (EHV4).
Veterinary microbiology    February 9, 2000   Volume 71, Issue 1-2 37-51 doi: 10.1016/s0378-1135(99)00162-5
van Maanen C, de Boer-Luijtze E, Terpstra C.A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submi...
Standardisation of muscular biopsy of gluteus medius in French trotters.
Equine veterinary journal. Supplement    February 5, 2000   Issue 30 342-344 doi: 10.1111/j.2042-3306.1999.tb05246.x
Valette JP, Barrey E, Jouglin M, Courouce A, Auvinet B, Flaux B.Morphometric measurements were taken from 41 French trotters of various ages and both sexes. Biopsy location was determined for the dorsal compartment as being one-third of the distance from the tuber sacrale to the tuber coxae and for the ventral compartment as being one-third of the distance from the tuber coxae to the caudal Cd1-Cd2 intervertebral joint. Ten horses were biopsied at these 2 sites at a sampling depth equal to half the total depth of the compartment as measured by ultrasonography. The percentage of slow and fast myosin heavy chain fibres was measured by using an immunoenzymati...
Expression of CRISP proteins in the male equine genital tract.
Journal of reproduction and fertility. Supplement    January 25, 2000   Volume 53 67-72 
Schambony A, Hess O, Gentzel M, Töpfer-Petersen E.Cysteine rich secretory proteins (CRISPs) have been detected immunochemically in the equine male genital tract. CRISPs are secretory products of the epididymis, the ampulla and the seminal vesicle. A particular feature of the horse is the abundance of CRISPs in seminal plasma. CRISPs can also be detected in extracts of testicular, epididymal and ejaculated spermatozoa in increasing amounts. Unlike other seminal plasma proteins, they cannot be removed completely from spermatozoa by high salt treatment. The remaining CRISP antigens are localized on the midpiece, and the postacrosomal and equator...
Experimental infection of ponies with Borrelia burgdorferi by exposure to Ixodid ticks.
Veterinary pathology    January 22, 2000   Volume 37, Issue 1 68-76 doi: 10.1354/vp.37-1-68
Chang YF, Novosol V, McDonough SP, Chang CF, Jacobson RH, Divers T, Quimby FW, Shin S, Lein DH.Seven specific-pathogen-free (SPF) ponies, 1-5 years old, were exposed to Borrelia burgdorferi-infected adult ticks while being treated with dexamethasone over 5 consecutive days. One SPF pony (pony No. 178) was first exposed to laboratory-reared nymphs without B. burgdorferi infection and 3 weeks later was exposed to B. burgdorferi-infected adult ticks with concurrent dexamethasone treatment for 5 consecutive days. Four uninfected ponies treated with dexamethasone, exposed to laboratory-reared ticks without B. burgdorferi infection served as uninfected controls. Clinical signs, bacteriologic ...
Analyses of TCRB rearrangements substantiate a profound deficit in recombination signal sequence joining in SCID foals: implications for the role of DNA-dependent protein kinase in V(D)J recombination.
Journal of immunology (Baltimore, Md. : 1950)    January 21, 2000   Volume 164, Issue 3 1416-1424 doi: 10.4049/jimmunol.164.3.1416
Shin EK, Rijkers T, Pastink A, Meek K.We reported previously that the genetic SCID disease observed in Arabian foals is explained by a defect in V(D)J recombination that profoundly affects both coding and signal end joining. As in C.B-17 SCID mice, the molecular defect in SCID foals is in the catalytic subunit of the DNA-dependent protein kinase (DNA-PKCS); however, in SCID mice, signal end resolution remains relatively intact. Moreover, recent reports indicate that mice that completely lack DNA-PKCS also generate signal joints at levels that are indistinguishable from those observed in C.B-17 SCID mice, eliminating the possibilit...
Complete genomic RNA sequence of western equine encephalitis virus and expression of the structural genes.
The Journal of general virology    January 21, 2000   Volume 81, Issue Pt 1 151-159 doi: 10.1099/0022-1317-81-1-151
Netolitzky DJ, Schmaltz FL, Parker MD, Rayner GA, Fisher GR, Trent DW, Bader DE, Nagata LP.The complete nucleotide sequence of the 71V-1658 strain of western equine encephalitis virus (WEE) was determined (minus 25 nucleotides from the 5' end). A 5' RACE reaction was used to sequence the 5' terminus from WEE strain CBA87. The deduced WEE genome was 11508 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. The nucleotide composition was 28% A, 25% C, 25% G and 22% U. Comparison with partial WEE sequences of strain 5614 (nsP2-nsP3 of the nonstructural region) and strain BFS1703 (26S structural region) revealed comparatively little variation; a total of 149 nucl...
Replication of equine herpesvirus type 1 in freshly isolated equine peripheral blood mononuclear cells and changes in susceptibility following mitogen stimulation.
The Journal of general virology    January 21, 2000   Volume 81, Issue Pt 1 21-25 doi: 10.1099/0022-1317-81-1-21
van Der Meulen KM, Nauwynck HJ, Bí¶®rt W, Pensaert MB.In the present study, the outcome of an inoculation of equine peripheral blood mononuclear cells (PBMC) with equine herpesvirus type 1 (EHV-1) was studied in vitro. Cytoplasmic and plasma membrane expression of viral antigens, intra- and extracellular virus titres, and plaque formation in co-culture were determined. EHV-1 replicated in monocytes, although in a highly restricted way. Viral antigens were found at maximum levels (8.7% of the monocytes) at 12 h post-infection. The infection was productive in 0.16% of the monocytes. The virus yield was 10(0.7) TCID(50) per productive cell. In a pop...
Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock.
Zentralblatt fur Veterinarmedizin. Reihe A    January 19, 2000   Volume 46, Issue 10 621-631 doi: 10.1046/j.1439-0442.1999.00256.x
Möstl E, Messmann S, Bagu E, Robia C, Palme R.After 14C-labelled cortisol infusion in ponies and pigs, faecal samples were collected. Extraction of 0.5 g faeces with 5 ml 80-90% methanol yielded the highest radioactivity in the supernatant. Most of the metabolites were ether soluble. After high performance liquid chromatography (HPLC), the presence of immunoreactive metabolites was demonstrated by measuring each HPLC fraction using enzyme immunoassays for cortisol, corticosterone and 11-oxoaetiocholanolone. Only the assay for 11-oxoaetiocholanolone revealed peaks with co-eluting radioactivity. For biological validation of the test system,...
Equine sarcoid tumour treated by autogenous tumour vaccine.
Anticancer research    January 12, 2000   Volume 19, Issue 4C 3367-3374 
Kinnunen RE, Tallberg T, Stenbäck H, Sarna S.Twenty-one horses with sarcoid tumours were treated by bio-immunotherapy using autogenous vaccines during 1991-1997. At operation the base of the tumour was left in the skin and autovaccines were made from extirpated tumour tissue by polymerization. The horses thus formed their own internal control group. One of 12 horses having a primary tumour, and four of 9 horses suffering recurrent tumours, prior to bio-immunotherapy, developed single recurrences. Four of these five horses suffering recurrence were treated anew, leading to remission. Disease-free intervals were longer for primary (P = 0.0...
Equid herpesvirus-induced immunosuppression is associated with lymphoid cells and not soluble circulating factors.
Viral immunology    January 12, 2000   Volume 12, Issue 4 313-321 doi: 10.1089/vim.1999.12.313
Hannant D, O'Neill T, Ostlund EN, Kydd JH, Hopkin PJ, Mumford JA.A paresis isolate of equid herpesvirus 1 (EHV1, Ab4/8) and a plaque-purified virus derived from it (EHV1, Ab4/13), induced long-term suppression of both mitogenic and antigen-specific lymphocyte proliferations in adult outbred ponies. Peripheral blood mononuclear cells (PBMC) taken from a pony after EHV1 infection suppressed the in vitro function of normal cells but serum did not. This showed that the observed immune suppression was associated with circulating PBMC and/or their products rather than circulating soluble factors such as antigen or immune complexes. The results suggested that prod...
[Validation of an indirect immunoenzyme assay for the detection of antibodies against Trypanosoma evansi in horses in Argentina].
Revue scientifique et technique (International Office of Epizootics)    January 11, 2000   Volume 19, Issue 3 810-818 
Monzón CM.An indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Trypanosoma evansi was evaluated using 90 different sera, obtained from naturally-infected horses. As negative controls, 218 sera from the T. evansi-free zone of Argentina, and 90 uninfected sera from the enzootic zone were used. The results of the ELISA were expressed in terms of percent positivity (PP) when compared with a positive primary reference serum, obtained from a horse experimentally-infected with T. evansi. The inter-assay coefficient of variation (CV), expressed as PP, was 44.7% for the negative con...
Kinetics of equine neutrophil elastase release and superoxide anion generation following secretagogue activation: a potential mechanism for antiproteinase inactivation.
Veterinary immunology and immunopathology    January 11, 2000   Volume 72, Issue 3-4 257-275 doi: 10.1016/s0165-2427(99)00144-0
Dagleish MP, Pemberton AD, Brazil TJ, McAleese SM, Miller HR, Scudamore CL.Man and horses both suffer from neutrophil mediated pulmonary diseases however there are striking species differences in the underlying pathology. In particular while pulmonary emphysema is a common pathological sequel to human respiratory disease it is not a major feature of the common equine neutrophil mediated condition, chronic obstructive pulmonary disease (COPD). The proposed reason for this difference is that equine neutrophils contain less elastase than equivalent human cells and therefore there is a reduced risk of excess and/or uninhibited elastase activity, which is considered the m...
Role of dendritic cell targeting in Venezuelan equine encephalitis virus pathogenesis.
Journal of virology    January 7, 2000   Volume 74, Issue 2 914-922 doi: 10.1128/jvi.74.2.914-922.2000
MacDonald GH, Johnston RE.The initial steps of Venezuelan equine encephalitis virus (VEE) spread from inoculation in the skin to the draining lymph node have been characterized. By using green fluorescent protein and immunocytochemistry, dendritic cells in the draining lymph node were determined to be the primary target of VEE infection in the first 48 h following inoculation. VEE viral replicon particles, which can undergo only one round of infection, identified Langerhans cells to be the initial set of cells infected by VEE directly following inoculation. These cells are resident dendritic cells in the skin, which mi...
Interactions between equine cyclin T1, Tat, and TAR are disrupted by a leucine-to-valine substitution found in human cyclin T1.
Journal of virology    January 7, 2000   Volume 74, Issue 2 892-898 doi: 10.1128/jvi.74.2.892-898.2000
Taube R, Fujinaga K, Irwin D, Wimmer J, Geyer M, Peterlin BM.Transcriptional transactivators (Tat) from human immunodeficiency and equine infectious anemia viruses (HIV and EIAV) interact with their transactivation response elements (TAR) to increase the rates of viral transcription. Whereas the human cyclin T1 is required for the binding of Tat to TAR from HIV, it is unknown how Tat from EIAV interacts with its TAR. Furthermore, Tat from EIAV functions in equine and canine cells but not in human cells. In this study, we present sequences of cyclins T1 from horse and dog and demonstrate that their N-terminal 300 residues rescue the transactivation of Ta...
Influence of age and parity on the distribution of cells expressing major histocompatibility complex class II, CD4, or CD8 molecules in the endometrium of mares during estrus.
American journal of veterinary research    January 6, 2000   Volume 60, Issue 12 1531-1535 
Tunón AM, Rodríguez-Martínez H, Nummijärvi A, Magnusson U.To evaluate effect of age and parity on distribution and number of cells expressing major histocompatibility complex (MHC) class II, CD4, or CD8 molecules in the endometrium of mares during estrus. Methods: 32 gynecologically healthy mares, categorized as young (3 to 8 years; n = 17) or old (9 to 16 years; 15) and nulliparous (n = 6), nulliparous embryo donors (16), or parous (10). Methods: Endometrial specimens collected from the uterine body and horns during estrus were stained by use of the avidin-biotin-peroxidase method, using monoclonal antibodies against equine MHC class II, CD4, and CD...
‘Cross-protection’ and ‘cross-reaction’ with equine influenza vaccines.
The Veterinary record    January 5, 2000   Volume 145, Issue 22 647 
Newton JR, Wood JL, Jessett D, Yates PJ, Mumford JA.No abstract available
Human granulocytic ehrlichiosis agent infection in a pony vaccinated with a Borrelia burgdorferi recombinant OspA vaccine and challenged by exposure to naturally infected ticks.
Clinical and diagnostic laboratory immunology    January 5, 2000   Volume 7, Issue 1 68-71 doi: 10.1128/CDLI.7.1.68-71.2000
Chang YF, McDonough SP, Chang CF, Shin KS, Yen W, Divers T.A pony was vaccinated with recombinant OspA vaccine (rOspA) and then exposed 3 months later to Borrelia burgdorferi-infected ticks (Ixodes scapularis) collected in Westchester County, N.Y. At 2 weeks after tick exposure, the pony developed a high fever (105 degrees F). Buffy coat smears showed that 20% of neutrophils contained ehrlichial inclusion bodies (morulae). Flunixin Meglumine (1 g daily) was given for 2 days, and the body temperature returned to normal. PCR for ehrlichial DNA was performed on blood samples for 10 consecutive days beginning when the pony was first febrile. This pony was...
Proceedings of the Seventh International Symposium on Equine Reproduction, July 12-17, 1998, Pretoria, South Africa.
Journal of reproduction and fertility. Supplement    January 1, 2000   Issue 56 3-734 
No abstract available
Ovarian secretion of inhibin in mares.
Journal of reproduction and fertility. Supplement    January 1, 2000   Issue 56 239-245 
Tanaka Y, Nagamine N, Nambo Y, Nagata S, Nagaoka K, Tsunoda N, Taniyama H, Yoshihara T, Oikawa M, Watanabe G, Taya K.In mares, circulating immunoreactive inhibin concentrations increase during the follicular phase and decrease at the start of the LH surge. Thereafter, sharp increases in circulating immunoreactive inhibin concentrations, the 'ovulatory increase', are observed during ovulation. In the present study, the cellular sources and molecular form of ovarian inhibin were investigated to determine the mechanism responsible for this unique ovulatory increase. Three sizes of ovarian follicles (small, 30 mm in diameter) were selected. Inhibin alpha-subunit was localized by immunohistochemistry to the gran...
Immunization with stallion lymphocytes for treatment of recurrent spontaneous abortion in thoroughbred mares.
Journal of reproduction and fertility. Supplement    January 1, 2000   Issue 56 645-650 
Mathias S, Allen WR.The incidence of recurrent spontaneous abortion within the human population in the Western world is low (2-5%) but significant, and a proportion of these pregnancy losses are thought to have an underlying immunological cause. Immunization of women who have a history of recurrent spontaneous abortion with lymphocytes isolated from their husband or a third party donor is one of several forms of immunotherapy used to treat the problem. Early pregnancy loss in Thoroughbred mares is also significant and, as in women, a small number of mares undergo repeated pregnancy losses. Two trials have been pe...
Equine trophoblast as an immunological target.
Journal of reproduction and fertility. Supplement    January 1, 2000   Issue 56 635-644 
Baker JM, Bamford AI, Carlson ML, Mcculloch CE, Antczak DF.A cytotoxic T-lymphocyte assay was used to determine whether equine chorionic girdle cells are susceptible to lysis by CD8+ cytotoxic T cells primed in vitro against allogeneic lymphocytes. Classical cytotoxic T-lymphocyte activity against fresh or cultured trophoblast targets was demonstrated using peripheral blood lymphocytes from nonpregnant mares. Lysis of allogeneic (horse) and xenogeneic (donkey) lymphocyte targets was used as a control for trophoblast lysis. The major histocompatibility complex (MHC) class I specificity of the cytotoxic T-lymphocyte reactions was determined using panels...
Immunolocalization of oxytocin and neurophysin in the mare uterus.
Journal of reproduction and fertility. Supplement    January 1, 2000   Issue 56 289-296 
Watson ED, Buckingham J, Björkstén T, Nikolakopoulos E.The presence of oxytocin in the mare uterus was determined by radioimmunoassay of tissue extracts and uterine lavage fluid, and by immunohistochemistry. Samples were obtained from mares during the oestrous cycle and on day 14 of pregnancy. Immunoreactive oxytocin was detected in extracts of endometrium and myometrium, and in uterine lavage fluid. In tissue sections, the luminal epithelium, the epithelium of the superficial, but not deep, glands, blood vessel walls and myometrium all stained positively for oxytocin, whereas only the luminal epithelium and epithelium of the superficial glands st...
Immunolocalization of a novel protein (P19) in the endometrium of fertile and subfertile mares.
Journal of reproduction and fertility. Supplement    January 1, 2000   Issue 56 593-599 
Stewart F, Gerstenberg C, Suire S, Allen WR.One of the major progesterone-dependent endometrial proteins in the mare is a novel 19 kDa lipocalin (P19). This protein is secreted by the endometrial glands and is readily detectable in uterine secretions during the luteal phase of the oestrous cycle and early pregnancy. The function of P19 is unknown, but since most lipocalins act as carriers of small hydrophobic molecules, it probably transports a maternal factor to the conceptus during pregnancy. In this study, a high titre antiserum raised against recombinant-derived P19 was used to detect by immunohistochemistry the protein in endometri...
Functional disturbances in the endometrium of barren mares: a histological and immunohistological study.
Journal of reproduction and fertility. Supplement    January 1, 2000   Issue 56 381-391 
Schoon HA, Wiegandt I, Schoon D, Aupperle H, Bartmann CP.Different types of endometrial maldifferentiation were investigated by histopathological (haematoxylin-eosin (HE), periodic acid Schiff-alcian blue and picro-sirius red staining) and immunohistological (oestrogen and progesterone receptors, Ki-67 antigen, intermediate filaments and laminin) analysis of endometrial biopsy specimens from mares (n=34), most of which had been barren for at least 1 year. Two major features were observed: (i) unequal differentiation, in which two functional stages are visible within a single biopsy sample: areas which are differentiated in accordance with the stage ...
Replication ability in vitro and in vivo of equine infectious anemia virus avirulent Japanese strain.
Virology    December 29, 1999   Volume 266, Issue 1 129-139 doi: 10.1006/viro.1999.0076
Zheng YH, Sentsui H, Sugita M, Nakaya T, Kishi M, Hagiwara K, Inoshima Y, Ishihara C, Kono Y, Lu JL, Ikuta K.An attenuated equine infectious anemia virus (EIAV), V26, was previously prepared by 50 passages of the Japanese virulent strain V70 in primary horse macrophage culture. The horses inoculated with this V26 virus were shown to raise neutralizing antibodies against V70 without any viremia. Here, we investigated the in vitro and in vivo replication ability of V26. Comparison of the long-terminal repeat (LTR) sequences between V26 and V70 revealed a large insertion within the LTR U3 hypervariable region of V26. V26 with the mutation in the LTR showed much higher promoter activity in vitro than V70...