Polyspecificity of antistreptococcal murine monoclonal antibodies and their implications in autoimmunity.

mAbs produced by immunization of BALB/c mice with Streptococcus pyogenes M type 5 membranes were further characterized for their reaction with S. pyogenes pep M5 protein and with autoantigens associated with human cell lines. mAbs 36.2.2 and 54.2.8 simultaneously reacted with M protein and a membrane protein(s) of S. pyogenes. When cell lines were mixed with 54.2.8, we saw nuclear fluorescence along with staining of the cytoskeleton. Subsequent experiments revealed that 54.2.8 was an anti-DNA antibody that reacted with DNA, poly(I), poly(dT), and weakly with cardiolipin. Its reactivity with the cytoskeleton could be blocked with anti-vimentin. On the other hand, 36.2.2 reacted with the cytoskeleton, sparing the nucleus, and was inhibited by the alpha helical proteins myosin, actin, and keratin. mAb 54.2.8 was inhibited with myosin, but not with actin and keratin. None of the antibodies studied were inhibited by collagen, and none of them were rheumatoid factors. The results imply that Group A streptococci can activate B cell clones against myosin, alpha helical proteins, or DNA, thereby contributing to the enhancement of autoantibody production.

Myosin: a link between streptococci and heart.

Murine monoclonal antibodies to Streptococcus pyogenes reacted with skeletal muscle myosin. High molecular weight proteins in extracts of human heart tissue that reacted with an antibody to S. pyogenes also reacted with a monoclonal antibody to ventricular myosin. Adsorption of the antibody to streptococci with S. pyogenes simultaneously removed reactivity of the antibody for either S. pyogenes or myosin. These results indicate that myosin shares immunodeterminants with a component of S. pyogenes.

Cytotoxic mAb from rheumatic carditis recognizes heart valves and laminin.

Anti-streptococcal antibodies cross-reactive with N-acetyl-betaD-glucosamine (GlcNAc) and myosin are present in the sera of patients with rheumatic fever (RF). However, their role in tissue injury is not clear. In this study, we show that anti-GlcNAc/anti-myosin mAb 3.B6 from a rheumatic carditis patient was cytotoxic for human endothelial cell lines and reacted with human valvular endothelium and underlying basement membrane. Reactivity of mAb 3.B6 with the valve was inhibited by human cardiac myosin > laminin > GlcNAc. The mAb 3.B6 epitopes were localized in fragments of human cardiac myosin, including heavy meromyosin (HMM), the S1 subfragment, and two light meromyosin (LMM) peptides containing amino acid sequences KEALISSLTRGKLTYTQQ (LMM 1) and SERVQLLHSQNTSLINQK (LMM 33). A novel feature of mAb 3.B6 was its reactivity with the extracellular matrix protein laminin, which may explain its reactivity with the valve surface. A laminin A-chain peptide (HTQNT) that includes homology to LMM33 inhibited the reactivity of mAb 3.B6 with human valve. These data support the hypothesis that cross-reactive antibodies in rheumatic carditis cause injury at the endothelium and underlying matrix of the valve.

Restricted immunoglobulin VH usage and VDJ combinations in the human response to Haemophilus influenzae type b capsular polysaccharide. Nucleotide sequences of monospecific anti-Haemophilus antibodies and polyspecific antibodies cross-reacting with self antigens.

To examine the human antibody repertoire generated against a biologically significant antigen we have obtained sequences of heavy chain variable region genes (IgVH) from 15 monoclonal antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). All VH segments are members of the VH3 family and 9 of 15 are members of the smaller VH3b subfamily. Restriction is evident by the shared use of certain VDJ joints in independent hybridomas from different subjects. Two hybridomas generated from the same subject demonstrate identical heavy chain variable region gene sequences but differ in isotype and rearrange alternative light chain variable region genes (IgVL), suggesting that in a normal immune response, a single pre-B cell clone may use different light chain rearrangements and give rise to progeny capable of reacting with antigen. Using a polymerase chain reaction assay optimized to detect base pair differences among VH genes we demonstrate that at least a portion of expressed anti-Hib PS VH genes have undergone somatic mutation. Anti-Hib PS heavy chain genes are homologous to VH segments encoding autoantibodies and two hybridomas secrete anti-Hib PS antibody that cross-reacts with self antigens (double-stranded DNA and single-stranded DNA). Comparison of VH regions of self-reactive and monospecific anti-Hib PS Ab demonstrates no consistent structural feature correlating with fine antigen specificity. These data demonstrate significant restriction in VH usage and VDJ recombination in the anti-Hib PS response and confirm that autoantibodies may be elicited during normal immune responses.

Autoimmunity against dopamine receptors in neuropsychiatric and movement disorders: a review of Sydenham chorea and beyond.

Antineuronal autoantibodies are associated with the involuntary movement disorder Sydenham chorea (SC) and paediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) which are characterized by the acute onset of tics and/or obsessive compulsive disorder (OCD). In SC and PANDAS, autoantibodies signal human neuronal cells and activate calcium calmodulin-dependent protein kinase II (CaMKII). Animal models immunized with group A streptococcal antigens demonstrate autoantibodies against dopamine receptors and concomitantly altered behaviours. Human monoclonal antibodies (mAbs) derived from SC target and signal the dopamine D2L (long) receptor (D2R). Antibodies against D2R were elevated over normal levels in SC and acute-onset PANDAS with small choreiform movements, but were not elevated over normal levels in PANDAS-like chronic tics and OCD. The expression of human SC-derived anti-D2R autoantibody V gene in B cells and serum of transgenic mice demonstrated that the human autoantibody targets dopaminergic neurones in the basal ganglia and other types of neurones in the cortex. Here, we review current evidence supporting the hypothesis that antineuronal antibodies, specifically against dopamine receptors, follow streptococcal exposures and may target dopamine receptors and alter central dopamine pathways leading to movement and neuropsychiatric disorders.

Molecular analysis of cross-reactive anti-myosin/anti-streptococcal mouse monoclonal antibodies.

Nucleotide sequences of VH- and VL-genes of anti-myosin/anti-streptococcal monoclonal antibodies (mAbs) were analyzed and compared with their highly detailed antigen binding reactivities. Antigen-specificities of the cross-reactive mAbs included myosin, streptococcal M-protein, actin, keratin, N-acetyl-beta-D-glucosamine, vimentin, DNA, tropomyosin, troponin, and laminin as previously described. After nucleotide sequence analysis, homology indicated that some of the V gene sequences aligned with antibodies recognizing gangliosides and blood group antigens glycophorin M and N. Therefore, mAb reactivity with gangliosides and glycophorin M and N was identified. The cross-reactive mAbs utilized a heterogeneous group of germline V-heavy genes comprised of nine J558-, four 7183- and two Q52-family VH-genes. Germline V-light genes utilized by the mAbs included six Vkappa4/5-, three Vkappa8-, two Vkappa10-, three Vkappa19- and one Vkappa23-family VL-genes. No preferential VH/VL-chains correlated with any of the 12 different antigen reactivities, even for mAbs with nearly identical cross-reactivities. However, we did find that the cross-reactive mAb germline genes within a V gene family shared more homology among themselves than with other germline genes within their V gene families, suggesting convergent mutation. Cross-reactive mAbs with the highest relative avidity for myosin were found in the VH7183 family which contained two cytotoxic mAbs. Antibodies with V gene sequences most homologous to those of our cross-reactive anti-myosin/anti-streptococcal mAbs had specificities for laminin, DNA, carbohydrates, or blood group antigens and were reported to cause autoimmune disease in mice.

Monoclonal antibodies cross-reactive with group A streptococci and normal and psoriatic human skin.

Infection with group A streptococci has been implicated as a factor capable of exacerbating psoriasis. In order to explore the possibility of cross-reactivity between streptococcal antigens and human skin in this phenomenon, skin from psoriatic patients and control subjects was reacted with 3 monoclonal antibodies against group A streptococci and antibody binding was estimated by the indirect immunofluorescence technique. Monoclonal antibody 54.2.8 stained the nuclei and cytoplasm of cells within the epidermis and epidermal appendages, as well as cells scattered throughout the dermis. In contrast, monoclonal antibodies 49.8.2 and 36.2.2 labeled the cytoplasm of epidermal cells and epidermal appendages but did not react with nuclei. No difference in the staining patterns of control skin and uninvolved skin from patients with psoriasis was observed. However, skin from psoriatic lesions contained large amounts of cross-reactive skin component(s). Sera from patients with guttate psoriasis did not react differently with normal or psoriatic skin when compared with normal sera. Western immunoblots of skin extracts demonstrated that monoclonal antibody 54.2.8 reacted with a family of proteins in the molecular weight range of 60-70K. The results indicate that component(s) in human skin share cross-reactive epitopes with group A streptococci. Immunologic cross-reactions between group A streptococci and human skin may play an important role in the exacerbation of certain skin disorders following streptococcal infections.

Immunological mimicry between retinal S-antigen and group A streptococcal M proteins.

Immunological mimicry between host and microbial proteins has been suggested as a potential mechanism in the development of uveitis in humans. In this study immunological crossreactivity between anti-streptococcal monoclonal antibodies (MAbs) and the human eye was investigated. In indirect immunofluorescence, we demonstrated novel immunological crossreactivity of two anti-streptococcal MAbs (27 and 112) with the rod outer (and inner) segments of the retina of the human eye. In further studies, retinal S-Ag, a uveitogenic protein in the rod outer (and inner) segments, was found to react with the anti-streptococcal MAbs. In addition, several uveitogenic peptides of S-Ag were recognized by the anti-streptococcal MAbs. In the ELISA and Western immunoblot, anti-S-Ag MAbs crossreacted with group A streptococci and the streptococcal M protein further demonstrating sites of antigenic similarity. Homology between the retinal S-Ag and streptococcal M protein was observed in amino acid sequences repeated in the B repeat region of the streptococcal M5 protein. These data show that retinal S-antigen has immunological similarities with streptococcal M protein, a major virulence determinant and strong bacterial cell surface antigen.

Response of an abdominal aortic thrombotic occlusion to local low-dose streptokinase therapy.

Local low-dose streptokinase infusion (5000 to 6000 IU/hr) as compared to systemic streptokinase infusion (loading dose 250,000 IU/hr, maintenance dose 100,000 IU/hr) successfully relieved a total thrombotic abdominal aortic occlusion. No complications of bleeding or distal embolization occurred during streptokinase therapy, which required only 8 days of hospitalization. When seen at 4 months after streptokinase infusion, the patient was still free of symptoms. When no immediate threat of ischemic limb loss exists, streptokinase may offer a promising therapeutic alternative to surgical intervention for patients who represent a poor operative risk.

Prevalence of sarcocysts in Florida black bears (Ursus americanus floridanus).

Sections of muscle from Florida black bears (Ursus americanus floridanus) collected in the state of Florida were observed for the presence of Sarcocystis sp. sarcocysts. Two of 132 (1.5%) black bears had muscle sections containing sarcocysts. One sarcocyst was observed per muscle section. The sarcocysts averaged 181.5 x 99.0 microm by light microscopy of hematoxylin and eosin-stained sections.

Determining the recipient site for a sensory island pedicle.

When performing a sensory island transfer for restoration of sensibility to an area of pinch, one must be accurate in determining the recipient site. We present a simple and precise means of determining the recipient site for a sensory island transfer by ink-blotting the areas of contact of the thumb and index finger.

Trombiculiasis in the Florida black bear.

We found trombiculid mite (Trombiculidae) infestations in 32 of 101 (32%) freeranging Florida black bears (Ursus americanus floridanus) live-captured or necropsied in Florida from January 1999 to April 2000. Prevalence of chigger infestation was greatest in June with no infestations seen October to March. Chigger infestations were recognized as accumulations of bright orange granular material usually associated with hair shafts. Mites were found in clusters of one to 102 (mean +/- SD = 8.5+/-19.5) and were distributed primarily over the ventral abdomen and thorax, inguinal and axillary regions, and proximal medial aspect of the extremities. Mites were identified as larval Eutrombicula splendens. Cutaneous lesions were seen in two of 32 (6%) infested bears.

Seroprevalence of selected disease agents from free-ranging black bears in Florida.

Sera obtained from 66 free-ranging Florida black bears (Ursus americanus floridanus) from three geographic areas of Florida (USA) between November 1993 and August 1995 were tested for antibodies to 13 disease agents. Antibody prevalences were 3 positive of 37 tested (8%) Coxiella burnetti, 37 of 66 (56%) Toxoplasma gondii, 3 of 61 (5%) bluetongue virus/epizootic hemorrhagic disease virus (BTV/EHDV), 4 of 66 (6%) canine adenovirus-type 1, 5 of 66 (8%) canine distemper virus (CDV), 10 of 62 (16%) canine parvovirus (CPV), 7 of 66 (11%) eastern equine encephalitis virus, 4 of 66 (6%) western equine encephalitis virus, 2 of 66 (3%) Venezuelan equine encephalitis virus, and 11 of 66 (17%) St. Louis encephalitis virus. No samples had serologic evidence of exposure to Brucella spp. (n = 37), Francisella tularensis (n = 40), or pseudorabies virus (n = 37). This is the first known published report of antibodies to BTV/EHDV, CDV, and CPV in black bears.

Vitamin A concentrations in serum and liver from Florida panthers.

Many of the anomalies and clinical signs afflicting the Florida panther (Felis concolor coryi) are suggestive of vitamin A deficiency. Our objectives in this study were to determine if a vitamin A deficiency exists in the free-ranging panther population and to determine if there are differences in vitamin A levels among various subgroups of free-ranging panthers. Retinol concentrations were used as an index to Vitamin A concentrations and were determined in serum and liver from free-ranging (serum, n = 45; liver, n = 22) and captive (serum, n = 9; liver, n = 2) juvenile and adult Florida panthers from southern peninsular Florida (USA), and in liver from free-ranging cougars (F. concolor subspp.) from Washington (USA) and Texas (USA) between November 1984 and March 1994. Combined juvenile (6- to 24-mo-old) and adult (> 24-mo-old) free-ranging Florida panthers had mean +/- SD serum retinol concentrations of 772.5 +/- 229 pmol/ml. Adult free-ranging Florida panthers had mean liver retinol concentrations of 4794.5 +/- 3747 nmol/g. Free-ranging nursing Florida panther kittens (age < 1 mo) had mean serum retinol concentrations of 397.9 +/- 69 pmol/ml. Among subgroups of free-ranging Florida panthers, females had higher corrected mean serum retinol concentrations than males and adult free-ranging Florida panthers had higher mean liver retinol concentrations than juveniles. Retinol concentrations in free-ranging Florida panthers did not differ significantly from those in captive panthers (liver and serum) or other free-ranging cougars (liver). Based on limited published values and our controls, a vitamin A deficiency could not be demonstrated in the Florida panther population nor were any subgroups or individuals considered deficient.

Pathogenicity of the hookworm, Ancylostoma pluridentatum, in a Florida panther (Felis concolor coryi) kitten.

We evaluated clinical signs and administered anthelmintic treatment to a wild-caught, captive Florida panther (Felis concolor coryi) kitten from Big Cypress National Preserve, Florida (USA) infected with the hookworm, Ancylostoma pluridentatum. Clinical signs observed included poor body condition, lethargy, and below normal red blood cell numbers, hemoglobin concentration, and packed cell volume, and elevated eosinophil numbers. In addition, a maximum of 936 Ancylostoma sp. eggs/g of feces were found on Day 11 of captivity. Following oral administration of 20 mg/kg pyrantel pamoate on Day 11, 26 A. pluridentatum were collected from the feces. Based on the resolution of clinical signs, cessation of egg shedding, and a return to normal hematologic values following anthelmintic treatment, we believe that infection with A. pluridentatum was the primary cause of the stressed conditions in the panther kitten.

Cryptorchidism and delayed testicular descent in Florida black bears.

Retained testes were found in 11 (16%) of 71 black bears (Ursus americanus) examined over a 3-year period in Florida (USA). Four of the 11 bears were older than one year and weighed more than 32 kg; therefore, they were considered to be cryptorchid. The remaining seven bears may have had delayed testicular descent due to their apparent normal immature development. This is the first known published report of the prevalence of cryptorchidism and apparently normal delayed testicular descent in a black bear population.

Atrial septal defects in Florida panthers.

Ostium secundum atrial septal defects (ASDs) were observed in six (3 M, 3 F) of 33 (20 M, 13 F) (18%) Florida panthers (Puma concolor coryi) necropsied by veterinary pathologists between 1985 and 1998. A seventh ASD was found in a female panther necropsied in the field and is included in the pathological description but not the prevalence of ASDs in Florida panthers. One panther (FP205) with severe ASD also had tricuspid valve dysplasia (TVD). Atrial septal defects and/or TVD are believed to have caused or contributed to the deaths of three (9%) Florida panthers in this study. Mean diameter +/- SD of ASDs was 9.0 +/- 4.7 mm (range 3 to 15 mm). Gross pathological changes attributed to ASDs/TVD in severely affected panthers (ASD > or = 10 mm) (n = 4) included mild right ventricular dilatation (n = 3) and hypertrophy (n = 2), mild to severe right atrial dilatation (n = 2), and acute pulmonary edema (n = 3). Panthers with mild ASDs (ASD < or = 5 mm) (n = 3) had no other detectable gross pathological changes associated with the ASDs. Histological examination of lungs of three panthers with severe ASDs revealed mild to moderate dilatation with fibrosis and smooth muscle atrophy of the tunica media of medium to large caliber arteries (n = 2), interstitial and/or pleural fibrosis (n = 2), perivascular fibrosis (n = 1), and acute to chronic edema (n = 3). Twenty-six necropsied panthers were examined one or more times while living; medical records were retrospectively evaluated. Antemortem radiographic, electrocardiographic, and echocardiographic examinations were performed on two panthers with severe ASDs (FP20 and FP205). Thoracic radiographic abnormalities in both included right heart enlargement, and in FP205 (severe ASD and TVD), mild pulmonary overperfusion. Electrocardiographic examination of FP205 revealed a right ventricular hypertrophy pattern, while FP205 had a normal electrocardiogram. Echocardiographic examination of FP20 revealed marked right atrial dilatation; a bubble contrast study indicated regurgitation across the tricuspid valve. Echocardiographic abnormalities in FP20 included right atrial and ventricular lilatation, atrial septal drop-out, and severe tricuspid regurgitation; non-selective angiography revealed significant left to right shunting across the ASD. All panthers with severe ASDs ausculted (n = 3) had systolic right or left-sided grade I-V/VI murmurs loudest at the heart base. All male panthers with ASDs (n = 3) (100%) and 9 of 17 (53%) male panthers without ASDs in this study were cryptorchid.

Vacuum-assisted closure versus saline-moistened gauze in the healing of postoperative diabetic foot wounds.

Diabetic foot wounds present a great challenge to wound care practitioners. The objective of this pilot study was to determine whether vacuum-assisted closure (V.A.C.) therapy would afford quicker wound resolution as compared to saline-moistened gauze in the treatment of postoperative diabetic foot wounds. Ten patients were randomized into either the experimental V.A.C. group or control saline gauze group. Included in the study were diabetic patients 18 to 75 years of age who had a nonhealing foot ulceration. Excluded were those patients with venous disease, coagulopathy, or those who had active infections not resolved by initial surgical debridement. All foot ulcers were surgically debrided prior to initiation of V.A.C. or gauze treatment. In the experimental group, V.A.C. dressings were applied in accordance with manufacturer's protocol for chronic wounds and changed every 48 hours. In the control group, saline gauze dressings were applied at the time of surgical debridement and changed twice a day thereafter. Measurements and photos were obtained to document wound progress. Main outcome measures included: 1) time to satisfactory healing (calculated from date of initial debridement to date of definitive closure, and 2) change in wound surface area (calculated from initial wound tracing to final tracing). Satisfactory healing in the V.A.C. group was achieved in 22.8 (+/- 17.4) days, compared to 42.8 (+/- 32.5) days in the control group. Surface area changes of 28.4% (+/- 24.3) average decrease in wound size in the V.A.C. group, compared to a 9.5% (+/- 16.9) average increase in the control group during measurement period.