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PubMed1994

Human parvovirus B19 serology with recombinant VP1 and VP2 antigens: diagnosis of acute infections by detecting B19-specific IgM and IgA antibodies.

BACKGROUND: The availability of immunoassays for the laboratory diagnosis of human parvovirus B19 (B19) infection, which is commonly associated with erythema infectiosum in children and arthropathy and arthralgia in adults has been hampered by the lack of native B19 antigen. The production of abundant supplies of recombinant (r) B19 proteins, through the cloning of the B19 genome into expression vectors, has led to a proliferation of assays for detecting B19-specific antibodies. OBJECTIVES: This study was undertaken to evaluate serological assays for detecting B19-specific IgM and IgA antibodies using rVP1 and rVP2 B19 viral coat proteins. Their suitability in the diagnosis of acute B19 infections and the incidence of non-specific reactivity were determined. STUDY DESIGN: A panel of sera consisting of B19-specific IgM-positive and -negative samples was tested for B19-specific IgM and IgA antibodies in an indirect IFA using rVP1 antigen. These samples and a further panel collected from patients with other virus infections and samples containing rheumatoid factor were tested for B19-specific IgM in an antibody-capture ELISA and an indirect ELISA, both of which utilized rVP2 antigen. RESULTS: Data from the two ELISAs using rVP2 antigen and the IFA with rVP1 antigen all showed significant correlation (P >/= 0.001) with a reference RIA using native B19 antigen. Non-specific reactions were observed with Paul-Bunnell-positive and rubella virus-specific IgM antibody-positive sera in the ELISAs but not in the IFA. B19-specific IgA antibodies were detected in all sera containing B19-specific IgM antibodies but were also found in a small number of sera collected from healthy blood donors with no history of recent B19 infection. CONCLUSION: This study demonstrates the usefulness of assays employing rVP1 and rVP2 B19 antigens for detecting B19-specific antibodies. The use of IgM-specific ELISAs allows the processing of large numbers of samples and the absence of non-specific reactivity in the IFA may indicate a role for this assay as a confirmatory test.

Journal Article↗
PubMed1996

Detection of respiratory viruses in nasopharyngeal secretions with immunofluorescence technique for multiplex screening-an evaluation of the Chemicon assay.

BACKGROUND: Antigen detection with immunofluorescence is an efficient method for diagnosis of respiratory tract infections, but has previously not allowed for simple screening of many respiratory viruses. Pools of monoclonal antibodies against various respiratory viruses are now available, and are potentially important tools for improvement of antigen detection in nasopharyngeal samples. OBJECTIVE: To evaluate the commercially available Chemicon immunofluorescence assay (IFA; respiratory viruses panel and identification kit), an indirect IFA containing a pool of monoclonal antibodies for screening for influenza A, B, respiratory syncytial virus (RSV), parainfluenza 1, 2, 3 and adenovirus, and the respective individual antibodies. STUDY DESIGN: Ninety-six frozen preparations from nasopharyngeal secretions or bronchoalveolar lavages were retrospectively examined with the assay, and the results compared with other IFAs for antigen detection and cell culture isolation obtained in the everyday routine. Nasopharyngeal preparations from 300 children with lower respiratory tract infections at Beijing Children's Hospital during the 1994-1995 winter season were also examined. RESULTS: The sensitivity of the Chemicon assay compared to the combined results of routine IFA and isolation was 89% and specificity 92%. If five identifications of RSV made with the Chemicon assay alone were regarded to be truly positive, the specificity was 100%. A viral etiology was identified in 105/280 (38%) evaluable samples drawn from the Chinese children (influenza A 20%, RSV 14%, adenovirus 3% and parainfluenza 1, 2 or 3, 7%). CONCLUSION: One problem with the Chemicon assay was that for around 4-13% of samples there was a non-specific staining in the screening assay, necessitating stainings for verification. Despite this, the assay is an excellent tool for identification of viral respiratory tract infections, giving an increased sensitivity compared to direct immunofluorescence assays.

Journal Article↗
PubMed2004

Estimation of diagnostic test characteristics and prevalence of Giardia duodenalis in dairy calves in Belgium using a Bayesian approach.

A Bayesian approach was used to determine both the test properties of three diagnostic test procedures and the prevalence of Giardia duodenalis in dairy calves in Belgium. A cross-sectional survey was conducted in the province of East Flanders, Belgium. Between September 2001 and December 2003, a total of 100 farms were visited and faecal samples were obtained rectally from 499 calves aged from newborn to 70 days. Because there is no gold standard for the diagnosis of a G. duodenalis infection in dairy calves, a subset of 235 samples obtained on the first 50 farms, was examined using three different assays: microscopical examination, an immunofluorescence assay (IFA) and an antigen detecting Elisa (ELISA). Based on the results of these three tests, Bayesian analysis indicated that the prevalence of G. duodenalis in dairy calves was 0.19 (95% Confidence Interval: 0.11-0.28) and that ELISA (Sensitivity (Se) 0.89 and Specificity (Sp): 0.90) and IFA (Se: 0.77 and Sp: 0.95) were both sensitive and specific diagnostic techniques, whereas microscopical examination was less sensitive (Se: 0.56 and Sp: 0.87). The proportion of positive farms was estimated as 0.42 (0.24-0.62). The prevalence and the cyst excretion in calves from different age categories were based on data obtained by IFA on all 499 samples. The prevalence was highest among four to five week old calves and remained high among older calves up to 10 weeks, but was lower among calves before the age of two weeks. The number of excreted cysts was estimated by IFA and ranged from 100 to 1,040,000 cysts per gram faeces, with a mean of 3516 cysts per gram faeces. The intensity of excretion peaked among four-week-old calves and remained high among calves up to the age of eight weeks. This is the first known study to use Bayesian analysis to estimate the prevalence of G. duodenalis in the faeces of dairy calves and to estimate test characteristics of diagnostic assays used for the detection of G. duodenalis.

Animals↗
PubMed2006

Reverse ELISA for IgG and IgM antibodies to detect Lassa virus infections in Africa.

BACKGROUND: Anti-Lassa antibodies are detected by indirect immunofluorescence assay (IFA) or by enzyme-immunoassay (ELISA). Both methods have problems to detect low amounts of specific antibodies. OBJECTIVES: We report here highly sensitive and specific reverse ELISAs to detect Lassa virus IgG and IgM antibodies. Due to the reverse techniques, serum samples could be applied at dilutions of 1:10 without increasing non-specific background reactions. STUDY DESIGN: For IgM antibody detection microtiter plates were coated with anti-IgM antibodies and for IgG antibody detection with rheumatoid factor (RF) (Sachers M, Emmerich P, Mohr H, Schmitz H. Simple detection of antibodies to different viruses using rheumatoid factor and enzyme-labelled antigen (ELA). J Virol Methods 1985;10:99-110). In both assays a tissue culture antigen was used in combination with a labeled anti-Lassa monoclonal antibody (Hufert FT, Ludke W, Schmitz H. Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies. Arch Virol 1989;106(3-4):201-12). RESULTS: The reverse ELISA turned out to detect virus-specific IgG and IgM antibody in all 20 samples of West African patients collected 2-8 weeks after onset of Lassa fever. Moreover, both IFA and reverse ELISA found IgG antibodies in 53 out of 643 samples of healthy West Africans (sensitivity of 100%). Six of the 643 samples were positive by reverse IgG ELISA only. Thus, the specificity compared to IIF was 99.0%, but it may be even higher, because compared to IFA the IgG ELISA was clearly more sensitive in detecting low antibody titers. CONCLUSIONS: In Ghana 3% seropositives were found by IFA, but 4% by the reverse ELISA. The reverse ELISAs can be performed with high sensitivity and specificity under field conditions in Africa.

Africa↗
PubMed2006

The RPS adeno detector for diagnosing adenoviral conjunctivitis.

PURPOSE: To compare the sensitivity, specificity, and accuracy of the RPS Adeno Detector (Rapid Pathogen Screening Inc., South Williamsport, PA) against both viral cell culture with confirmatory immunofluorescence staining (CC-IFA) and the polymerase chain reaction (PCR) for diagnosing adenoviral conjunctivitis. DESIGN: Prospective, nonrandomized, masked, multicenter clinical trial. PARTICIPANTS: One hundred eighty-six consecutive patients from 5 clinical centers seeking treatment within 1 week of developing a red eye and thought to have acute conjunctivitis. METHODS: The RPS Adeno Detector is a 10-minute in-office lateral flow immunoassay. Patients were tested with the RPS Adeno Detector, CC-IFA, and PCR to detect the presence of adenovirus. MAIN OUTCOME MEASURES: The sensitivity, specificity, and accuracy of the RPS Adeno Detector were assessed for identifying cases of adenoviral conjunctivitis. RESULTS: Compared with CC-IFA, the RPS Adeno Detector was 88% sensitive and 91% specific at detecting adenoviral conjunctivitis. Using PCR as a reference method, the sensitivity of the RPS Adeno Detector increased to 89% and the specificity increased to 94%. Compared with PCR, CC-IFA was found to be 91% as sensitive and 100% as specific. CONCLUSIONS: The RPS Adeno Detector demonstrated sufficient sensitivity and specificity to be used in the physician's office for the detection of adenoviral conjunctivitis.

Acute Disease↗
PubMed2004

What has happened in norway after the ban of avoparcin? Consumption of antimicrobials by poultry.

When avoparcin was prohibited for use as feed additive in poultry in Norway on 31 May 1995, an increased incidence of Clostridium perfringens-associated necrotic enteritis (NE) and an increase in the use of antibacterial (AB) drug therapy in meat-type poultry was expected. The consumption of AB drugs for use against NE in poultry in the period 1990-2001 was investigated by use of sales statistics at the drug-wholesaler level. Defined daily dose (DDD) per kg live weight poultry was the unit of measurement for drug use (to correct for differences in the dosages). Sales figures of the AB drugs were converted to number of DDDpoultry sold for the numbers of broilers at risk (broilers were 97% of the slaughter poultry). Estimated annual percentages of the broilers treated against NE increased abruptly after the avoparcin ban--but in 1996, this figure declined to the same level as before the ban and has remained at that low level since then. In November 1995, narasin was approved temporarily as an ionophore feed additive (IFA) in broilers. The usage patterns of IFAs in broilers were measured as the weight of feed to which an IFA was added per broiler chicken produced. In 1996-2001, the IFAs used in broilers were predominantly narasin. We note that the temporary increase in NE after the avoparcin ban coincide with the period before narasin became available. The increase in the consumption of AB drugs for the treatment of NE in poultry following the avoparcin ban has been negligible.

Animal Feed↗
PubMed2005

Development and evaluation of a flow cytometry microsphere assay to detect anti-histone antibody in dogs.

Anti-nuclear antibody (ANA) is one of the diagnostic parameters that support a diagnosis of autoimmune disorders in humans, dogs, and horses, particularly the condition systemic lupus erythematosus (SLE). The most commonly used method for detecting ANA in canine serum is the indirect immunofluorescence antibody assay (IFA) that detects dog IgG with reactivity towards mammalian cell nuclei. Interpretation of the IFA results is very subjective and dependent on the source of tissue/cellular substrate. We have developed a flow cytometry based assay to detect canine serum antibodies specific to histones. Histones were chosen as the target antigen because these nuclear proteins are the most common nuclear substrate for ANA in dogs with SLE. Microsphere beads were coated with histones and incubated with canine sera. Bound anti-histone antibodies were detected by FITC-conjugated rabbit F(ab')2 anti-dog IgG. Sera from four groups of dogs (47 dogs total) were tested for anti-histone antibodies and compared with the traditional IFA assay. The groups included 15 healthy dogs, 15 dogs with noninflammatory diseases, 9 dogs with polyarthritis and positive ANA, and 8 German shepherds with perianal fistulas. The microsphere assay results indicated that only one dog in the noninflammatory group and four out of nine dogs in the polyarthritis group had mean fluorescent intensity values above our established cut-off (defined as 2 S.D. above the mean of healthy controls). There was moderate agreement between the anti-histone assay and the traditional ANA (kappa statistic=0.54). Absorption of ANA positive serum with total histones dramatically diminished the fluorescent signal detected by flow cytometry and the speckled nuclear pattern observed by IFA, whereas preabsorption did not change the diffuse nuclear staining pattern. These findings indicate that the anti-histone assay will not replace the ANA test and that other nuclear proteins, such as ribonucleoproteins may contribute to the diffuse ANA patterns.

Animals↗
PubMed2006

Evaluation of an ELISA rapid device for the serological diagnosis of Leishmania infantum infection in dog as compared with immunofluorescence assay and Western blot.

In this study we compared a commercial enzyme linked immunosorbent assay (ELISA) rapid test (Snap CLATK Canine Leishmania Antibody Test Kit, IDEXX-Snap) with indirect immunofluorescence assay (IFA) and Western blot (WB) for the detection of Leishmania infantum antibodies in dogs. In total sera from 234 dogs were collected: 59 positives and 51 doubtful sera (IFA 1:40-1:80) from an L. infantum endemic area and 124 negative sera from a non-endemic area were tested. To evaluate the Snap CLATK's performances on whole blood, blood in EDTA and sera from 37 dogs were tested in parallel with Snap CLATK. Snap CLATK sensitivity and specificity compared to IFA were 91.1% and 99.2%, while compared to WB were 93.4% and 98.3%, respectively. When IFA doubtful sera (titers of 1:40 or 1:80) were tested Snap CLATK, using WB as reference, sensitivity and specificity were 90.9% and 100%, respectively. Moreover, a complete concordance was observed when Snap CLATK rapid assay was carried out on whole blood or sera from 37 dogs. The Snap CLATK has demonstrated simplicity and performance and can be considered a quick and reliable alternative for the diagnosis of L. infantum infection in dogs.

Animals↗
PubMed1986

Seasonal variation in antigens of the imported fire ant Solenopsis invicta.

Eight sequentially collected lots of aqueous extracts of imported fire ant (IFA) front end and abdominal end segments were assayed for phospholipase A (PLA), N-acetyl-beta-glucosaminidase (NAG), and hyaluronidase. Relative potency of each extract lot and pooled venom was measured by RAST inhibition against a venom standard. More than a hundredfold difference in PLA activity was observed. Early summer collections had the highest activity. The May to June collection had more than twice the PLA activity of the next most potent lot. Discordancy in enzyme patterns was noted only in AE extract. NAG levels peaked earlier in the spring and summer and fluctuated less widely. Front end extract had lower activity levels for both enzymes, with no seasonal fluctuation in NAG and a single elevation in PLA activity in the April to May collection. RAST inhibition varied directly with PLA activity (p less than .05) but not with NAG nor hyaluronidase activities. Fifty-one percent of systemic allergic reactions to IFA stings occurred in summer, and 19% occurred in spring. A reported demographic survey demonstrated a higher incidence of IFA stings in the spring (39.9%) with a lower attack rate in the summer (31.9%). These findings suggest that the rate of systemic reactions to stings of the IFA may be related to seasonal variations in allergenic potency, as measured by PLA and RAST inhibition, rather than the sting attack rate.

Acetylglucosaminidase↗
PubMed1996

Prevalence of Kaposi's sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, may be the infectious cause of KS. Its prevalence in the general population, on the basis of detection of the virus genome, is controversial. To investigate the seroprevalence, we measured antibodies to a recombinant capsid-related (lytic cycle) KSHV antigen and a latent antigen complex. METHODS: We selected potentially immunoreactive capsid-related proteins of KSHV by expressing them as recombinant proteins and testing them in western blot assays. We used a truncated recombinant protein encoded by KSHV open reading frame 65 (orf 65) to develop a diagnostic enzyme-linked immunosorbent assay (ELISA) and tested sera from HIV-infected individuals with KS, HIV-uninfected patients with "classic" KS, other HIV risk groups, and blood donors. We also compared the antibody response to this capsid-related protein to the response to latent antigen(s) in an immunofluorescence assay. FINDINGS: 77/92 (84%) sera from KS patients reacted with the KSHV orf 65 protein and 84/103 (81.5%) reacted with KSHV latent antigen(s). The dominant immunogenic region of orf 65 is within the carboxyterminal 80 aminoacids, a region with little sequence similarity to the related Epstein-Barr virus, suggesting that orf 65 is a KSHV specific antigen. Only three sera from patients with haemophilia (1/84) or from intravenous drug users (2/63) had KSHV specific antibodies in the orf 65 assay whereas none of these sera reacted with latent antigen. Antibodies to KSHV were also infrequently found in UK and US blood donors by either assay (UK, 3/174 with orf 65 and 4/150 with latent antigen; US, 6/117 with orf 65 and 0/117 with latent antigen). They were more common among HIV-infected gay men without KS (5/16 by orf 65 ELISA, 10/33 by IFA), HIV-uninfected STD clinic attenders (14/166 by IFA), and Ugandan HIV-uninfected controls (6/17 by orf 65 ELISA, 9/17 by IFA). Antibody reactivity to the orf 65 protein (ELISA) and to latent antigen(s) (IFA) was concordant in 89% of 462 sera tested but reactive blood donor sera were discordant in both assays. Four AIDS-KS sera were unreactive in both assays. INTERPRETATION: The distribution of antibodies to both a capsid-related recombinant protein and latent antigen(s) of KSHV strongly supports the view that infection with this virus is largely confined to individuals with, or at increased risk for, KS. However, infection with KSHV does occur, rarely, in the general UK and US population and is more common in Uganda. Antibodies to latent antigen(s) or to orf 65 encoded capsid protein will not detect all cases of KSHV infection, and a combination of several antigens will probably be required for accurate screening and confirmatory assays.

Adult↗
PubMed1998

Antibody and cytokine responses in efferent lymph following vaccination with different adjuvants.

The cannulated efferent lymph node in sheep was used to examine the effect of different adjuvants on the antibody and cytokine responses following sub-cutaneous vaccination with a recombinant Taenia ovis antigen (45 W). Vaccination with Quil A elicited relatively higher levels of IgM than did IFA or Al(OH)3. In general, 45 W specific IgG1 and IgG2 titres were higher and maintained for longer periods of time in lymph from sheep vaccinated with IFA and lower and shorter lived in animals which received the Al(OH)3 based vaccine. Interferon-gamma was present within one day in efferent lymph from all sheep which received the Quil A formulation and in only one of the three sheep that received the IFA formulation. GM-CSF was only detected in lymph from sheep vaccinated with the IFA formulation. IL-8 was present in lymph prior to vaccination and only animals which received the Quil A formulation had increased levels of IL-8 after vaccination. Neither of the inflammatory cytokines IL-1 beta and TNF alpha were detected in efferent lymph from any animals in this study. This paper highlights the potential of the lymphatic cannulation model for investigations of the in vivo action of adjuvants.

Adjuvants, Immunologic↗
PubMed2001

Evaluation of a recombinant line blot for diagnosis of Epstein-Barr Virus compared with ELISA, using immunofluorescence as reference method.

A commercial line blot using recombinant antigens was compared with a commercial ELISA and 'in-house' IFA (reference test). Two panels were evaluated: Panel A was selected to distinguish between primary infections (89), past infections (20) and seronegatives (8) in immunocompetent individuals. In panel B, patients with a high number of reactivations were included: immunosuppressed patients (37), lymphoma (19), nasopharyngeal carcinoma (10), chronic fatigue syndrome (14). Blood donors (43) and cross-reactive sera (29) were added as controls. Line blot and IFA were concordant in 94% of primary infections, 100% of seronegatives and 100% of past infections, similar to ELISA. Results differed significantly with regard to reactivations. When compared with IFA, the incidence of reactivations was overestimated by the blot, 24 and 58% in blood donors and cross-reactive sera, respectively. ELISA showed a similar problems with 21 and 34% indeterminate results, respectively. The line blot is easy to carry out, has a good concordance with the reference IFA for primary infections, and is, therefore, a sufficient choice for distinguishing primary infection from seronegative and past infection. EBV reactivation assessment will require other methods such as EBV viral load.

Acute Disease↗
PubMed2002

Mucosal immune responses following oral immunization with rotavirus antigens encapsulated in alginate microspheres.

Availability of effective oral vaccine delivery vehicles should contribute to the success of oral immunization in domestic animals. To achieve this goal, we evaluated alginate microspheres for their capacity to induce mucosal immune responses following oral and enteric immunizations. Mice were immunized with either live porcine rotavirus (PRV) or its recombinant VP6 protein, encapsulated in alginate microspheres or unencapsulated. VP6-specific IgG (but no IgA) antibodies were detected in the sera of mice after a single intraperitoneal (i.p.) immunization with either VP6 in Incomplete Freund's adjuvant (VP6-IFA), VP6 in alginate microspheres (VP6-MS) or with live PRV in incomplete Freund's adjuvant (PRV-IFA). In contrast, VP6-specific IgA (but no IgG) was detected in culture supernatants of mesenteric lymph nodes from mice immunized i.p. with either VP6-IFA or with PRV-IFA. Oral immunization with VP6-MS induced the highest level of VP6-specific fecal IgA antibody, similar to responses induced by oral immunization with live PRV. Furthermore, the VP6-specific fecal IgA could be boosted by a secondary i.p. immunization with VP6. Further experiments were performed in a sheep intestinal 'loop' model to evaluate uptake of microspheres by Peyer's patches. Microspheres containing colloidal carbon were specifically bound and transported by follicle-associated epithelium of Peyer's patches. Additionally, mucosal immune responses were detected following enteric immunization with porcine serum albumin (PSA) encapsulated in alginate microspheres. Our results confirm that alginate microspheres are an effective oral delivery vehicle for protein antigens and intestinal IgA antibody responses are induced by antigens encapsulated in alginate microspheres without any additional mucosal adjuvant. These investigations confirm that alginate microspheres have the potential as an effective delivery vehicle for oral immunization of ruminants.

Administration, Oral↗
PubMed1987

Early detection and persistence of antibodies to Borrelia burgdorferi in persons with Lyme disease.

An indirect immunofluorescence antibody (IFA) test and an enzyme-linked immunosorbent assay (ELISA) were used during 1984 to detect total immunoglobulins (Ig) or IgM and IgG antibodies to Borrelia burgdorferi. Of the 139 serum samples tested by IFA, representing 110 persons with erythema chronicum migrans (ECM), 84 (60%) had either or both class-specific immunoglobulins. Serologic confirmation was made for 64 (58%) patients; serum specimens from 54 (49%) of these had elevated levels of IgM antibodies. These immunoglobulins were usually present within 14 days after diagnosis of ECM and persisted for at least 30 days. Significant titers of IgG antibodies were recorded for 54 serum samples and remained elevated for at least 19 weeks after ECM was first observed. When utilizing polyvalent (Ig) conjugates, ELISA procedures verified antibody presence in 80 of the 84 serum samples positive by IFA. Similarly, samples negative in IFA tests were non-reactive by ELISA. Additional tests of 40 control samples, obtained from persons without Lyme disease, were also uniformly negative. Both methods were suitable for confirmation of Lyme disease infections.

Antibodies, Bacterial↗
PubMed1987

Fatal Legionella pneumonia: retrospective examination of lung tissue using direct and indirect fluorescent-antibody methods.

Lung tissue sections of patients who died of pneumonia within one year were screened retrospectively for Legionnaires' disease bacteria using simultaneously a direct fluorescent antibody test (DFA) and an indirect fluorescent antibody system (IFA). The IFA has shown to provide a rapid screen for the increasing number of known Legionella species and serogroups recently. In 10/168 (6%) of our cases legionellae were the pneumonia causing agents detected with both methods. Although we should have been able to detect 25 serological variants of Legionella species and serogroups with the IFA, we found Legionella pneumophila serogroup 1 only. The IFA test has proven to be a reliable means for the screening of lung tissue sections.

Aged↗
PubMed1996

Induction of sustained and elevated immune responses to weakly immunogenic synthetic malarial peptides by encapsulation in biodegradable polymer microspheres.

Biodegradable microspheres (MS) based on poly(D,L-lactide) and poly(D,L-lactide-coglycolide) have the capacity to release encapsulated antigens over defined lengths of time depending on their composition and to elicit and sustain strong and long-lasting immune responses to protein antigens. In the present study, two synthetic multiple antigenic peptides (MAP), P30B2 and (NANP)6P2P30, were incorporated into MS of different compositions. P30B2 and (NANP)6P2P30 are composed of one or two universal T helper epitopes from tetanus toxin, 947-967 (P30) and 830-843 (P2), and of a B cell epitope derived from the repeat sequence of Plasmodium berghei or Plasmodium falciparum, respectively. BALB/c mice were immunized with these two peptides in different formulations, including individual MS or mixtures of MS with various release properties, Incomplete Freund's adjuvant (IFA) or as soluble peptides. MS formulations elicited strong and sustained proliferative and antibody responses comparable to those obtained with the IFA preparations. Furthermore, MS formulations induced specific isotype/ subclass antibodies similar to those induced by IFA. No significant augmentation of total serum IgE was detected during this study. In addition, a boosting effect was obtained when the immunized mice were reinjected with a small antigen dose in IFA several months later. These results indicate that biodegradable MS may be a suitable vaccine delivery system/adjuvant not only for protein antigens but also for weakly immunogenic synthetic peptides.

Amino Acid Sequence↗
PubMed1997

Myofibroblasts and arteriolar sclerosis in human diabetic nephropathy.

We examined the biopsy specimens of 62 patients with diabetic nephropathy to establish whether the myofibroblast (MF) has a role in progressive interstitial fibrosis and to ascertain whether a relationship existed between MF activity and severity of arteriolosclerosis. MF were identified by morphology and alpha smooth muscle actin (alpha SMA) immunostaining. Analysis of vascular injury was performed by counting the number of interstitial arterioles after staining endothelial cells with von Willebrand factor (VWF) antibody. Arteriosclerosis was quantified by using a computer-aided image analyzer to measure the arteriolar wall surface and total arteriolar surface area, and the ratio of wall to total surface area was expressed as the index of arteriosclerosis (IA). Fractional area of interstitium (IFA), alpha SMA, and collagen III (Coll III) were quantitated by point counting. Results were related to structural and functional parameters using rank correlation coefficients. There was a strong correlation between IFA and Coll III staining (r = 0.83; P < 0.001). The alpha SMA staining correlated with IFA (r = 0.56; P < 0.001) and Coll III (r = 0.47; P < 0.001), and there were significant correlations between alpha SMA and total urinary protein (r = 0.47; P < 0.001), renal function (plasma creatinine) at time of biopsy (r = 0.51; P < 0.001), and the percent change in plasma creatinine after 4 years (delta Cr) (r = 0.37; P = 0.01). The IA correlated significantly with Coll III (r = 0.29; P = 0.02), glomerular filtration rate (GFR) (r = 0.39; P = 0.008), and creatinine (r = 0.33; P = 0.01), but no correlation was observed between alpha SMA and IA (r = 0.16; P = 0.23) or IA and delta Cr (r = -0.04; P = 0.6). Strong correlations could be shown between arteriolar density, IFA (r = 0.75; P < 0.001), alpha SMA (r = -0.36; P = 0.034), and Coll III (r = -0.66; P < 0.0001). The MF appears to have a significant role in the progression of diabetic nephropathy. Ischemia secondary to arteriosclerosis may contribute to interstitial fibrosis through fibroblast modulation into MF.

Adult↗
PubMed2001

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.

Animals↗