Hussni O. Mohammed

Insulin-like growth factor-I enhances cell-based repair of articular cartilage

Composites of chondrocytes and polymerised fibrin were supplemented with insulin-like growth factor-I (IGF-I) during the arthroscopic repair of full-thickness cartilage defects in a model of extensive loss of cartilage in horses. Repairs facilitated with IGF-I and chondrocyte-fibrin composites, or control defects treated with chondrocyte-fibrin composites alone, were compared before death by the clinical appearance and repeated analysis of synovial fluid, and at termination eight months after surgery by tissue morphology, collagen typing, and biochemical assays. The structure of cartilage was evaluated histologically by Toluidine Blue reaction and collagen type-I and type-II in situ hybridisation and immunohistochemistry. Repair tissue was biochemically evaluated by DNA assay, proteoglycan quantitation and characterisation, assessment of collagen by reverse-phase high-performance liquid chromatography, and collagen typing using cyanogen bromide digestion and peptide separation by polyacrylamide gel electrophoresis. The results at eight months showed that the addition of IGF-I to chondrocyte grafts enhanced chondrogenesis in cartilage defects, including incorporation into surrounding cartilage. Gross filling of defects was improved, and the tissue contained a higher proportion of cells producing type-II collagen. Measurements of collagen type II showed improved levels in IGF-I-treated defects, supporting in situ hybridisation and immunohistochemical assessments of the defects. IGF-I improves the repair capabilities of chondrocyte-fibrin grafts in large full-thickness repair models.

Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans.

ABSTRACT A clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein A of 130 kDa (LigA) from Leptospira interrogans serovar pomona type kennewicki was isolated by screening a genomic DNA library with serum from a mare that had recently aborted due to leptospiral infection. LigA is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats. A search of the NCBI database found that homology of the LigA repeat region was limited to an immunoglobulin-like domain of the bacterial intimin binding protein of Escherichia coli, the cell adhesion domain of Clostridium acetobutylicum, and the invasin of Yersinia pestis. Secondary structure prediction analysis indicates that LigA consists mostly of beta sheets with a few alpha-helical regions. No LigA was detectable by immunoblot analysis of lysates of the leptospires grown in vitro at 30°C or when cultures were shifted to 37°C. Strikingly, immunohistochemistry on kidney from leptospira-infected hamsters demonstrated LigA expression. These findings suggest that LigA is specifically induced only in vivo. Sera from horses, which aborted as a result of natural Leptospira infection, strongly recognize LigA. LigA is the first leptospiral protein described to have 12 tandem repeats and is also the first to be expressed only during infection. Thus, LigA may have value in serodiagnosis or as a protective immunogen in novel vaccines.

Prevalence of Giardia sp., Cryptosporidium parvum and Cryptosporidium muris (C. andersoni) in 109 dairy herds in five counties of southeastern New York

Abstract A cross-sectional study was undertaken to determine the prevalence of Giardia sp. (G. duodenalis group), Cryptosporidium parvum and Cryptosporidium muris (C. andersoni) in dairy cattle in three different age groups, and to evaluate the association of age and season with prevalence. One hundred and nine dairy farms, from a total of 212 farms, in five counties of southeastern New York volunteered to participate. On these farms, 2943 fecal samples were collected from three defined age groups. The farms were randomly assigned for sampling within the four seasons of the year. Each farm was visited once during the study period from March 1993 to June 1994 to collect fecal samples. Demographic data on the study population was collected at the time of sampling by interviewing the farm owner or manager. At collection, fecal samples were scored as diarrheic or non-diarrheic, and each condition was later related to positive or negative infection with these parasites. Fecal samples were processed using a quantitative centrifugation concentration flotation technique and enumerated using bright field and phase contrast microscopy. In this study, the overall population prevalence for Giardia sp. was 8.9%; C. parvum, 0.9%; and C. muris, 1.1%. When considering animals most at the risk of infection (those younger than 6 months of age) Giardia sp. and C. parvum was found in 20.1 and 2.4% of the animals, respectively. Giardia sp. and C. muris were found in all age groups. There was no significant seasonal pattern of infection for any of these parasites.

The Anti-inflammatory and Matrix Restorative Mechanisms of Platelet-Rich Plasma in Osteoarthritis

Background: Intra-articular (IA) treatment with platelet-rich plasma (PRP) for osteoarthritis (OA) results in improved patient-reported pain and function scores. Purpose: To measure the effects of PRP and high molecular weight hyaluronan (HA) on the expression of anabolic and catabolic genes and on the secretion of nociceptive and inflammatory mediators from OA cartilage and synoviocytes. Study Design: Controlled laboratory study. Methods: Synovium and cartilage harvested from patients undergoing total knee arthroplasty were co-cultured with media of PRP or HA. Tumor necrosis factor–α (TNF-α), interleukin-6 (IL-6), and IL-1β were measured in the media by enzyme-linked immunosorbent assay. Hyaluronan synthase–2 (HAS-2), matrix metalloproteinase–1 (MMP-1), MMP-13, and TNF-α genes were measured in synoviocytes by reverse transcription polymerase chain reaction (RT-PCR). Collagen type I α1 (COL1A1), COL2A1, aggrecan (ACAN), and MMP-13 gene expression were measured in cartilage by quantitative RT-PCR. Results: Media TNF-α concentration was decreased in PRP and HA compared with control cultures (PRP = 6.94 pg/mL, HA = 6.39 pg/mL, control = 9.70 pg/mL; P ≤ .05). Media IL-6 concentration was decreased in HA compared with PRP and control (HA = 5027 pg/mL, PRP = 5899 pg/mL, control = 5613 pg/mL; P ≤ .05). Media IL-1β was below detectable concentrations (<0.1 pg/mL) in all samples. Synoviocyte MMP-13 expression was decreased in PRP compared with HA and control (PRP = 10.1, HA = 12.8, control = 13.5; P ≤ .05). Synoviocyte HAS-2 expression was increased in PRP compared with HA and control (PRP = 12.1, HA = 9.8, control = 8.7; P ≤ .05). Cartilage ACAN expression was increased in PRP compared with HA, but neither was different from control (PRP = 8.8, HA = 7.7, control = 7.6; P ≤ .05); COL1A1 expression was increased in HA compared with PRP, but neither was different from control (PRP = 14.9, HA = 13.5, control = 12.9; P ≤ .05). Neither platelet nor leukocyte concentration had a significant effect on outcome measurements (gene or protein expression data) in cartilage or synoviocytes (P > .05). Conclusion: Both PRP and HA treatments of OA joint tissues result in decreased catabolism, but PRP treatment also resulted in a significant reduction of MMP-13, an increase in HAS-2 expression in synoviocytes, and an increase in cartilage synthetic activity compared with HA. These results indicate that PRP acts to stimulate endogenous HA production and decrease cartilage catabolism. Platelet-rich plasma showed similar effects as HA in the suppression of inflammatory mediator concentration and expression of their genes in synoviocytes and cartilage. Clinical Relevance: The antinociceptive and anti-inflammatory activities of PRP support its use in OA joints to reduce pain and modulate the disease process. This study supports further clinical investigations of IA PRP for the treatment of OA.

Increasing Platelet Concentrations in Leukocyte-Reduced Platelet-Rich Plasma Decrease Collagen Gene Synthesis in Tendons

Background: Platelet-rich plasma (PRP) is used for the treatment of tendinopathy. There are numerous PRP preparations, and the optimal combination of platelets and leukocytes is not known. Hypothesis: Within leukocyte-reduced PRP (lrPRP), there is a plateau effect of platelet concentration, with increasing platelet concentrations being detrimental to extracellular matrix synthesis. Study Design: Controlled laboratory study. Methods: Different formulations of lrPRP with respect to the platelet:leukocyte ratio were generated from venous blood of 8 horses. Explants of the superficial digital flexor tendon were cultured in lrPRP products for 96 hours. Platelet-derived growth factor–BB (PDGF-BB), tumor necrosis factor−α (TNF-α), transforming growth factor–β1 (TGF-β1), and interleukin-1β (IL-1β) concentrations were determined in the media by enzyme-linked immunosorbent assay. Gene expression in tendon tissue for collagen type I and III (COL1A1 and COL3A1, respectively), matrix metalloproteinase−3 and −13 (MMP-3 and MMP-13, respectively), cartilage oligomeric matrix protein (COMP), and IL-1β was determined. Data were divided into 3 groups of lrPRP based on the ratio of platelets:leukocytes and evaluated to determine the effect of platelet concentration. Results: Complete blood counts verified leukocyte reduction and platelet enrichment in all PRP preparations. In the lrPRP preparation, the anabolic growth factors PDGF-BB and TGF-β1 were increased with increasing platelet concentrations, and the catabolic cytokine IL-1β was decreased with increasing platelet concentrations. Increasing the platelet concentration resulted in a significant reduction in COL1A1 and COL3A1 synthesis in tendons. Conclusion: Increasing the platelet concentration within lrPRP preparations results in the delivery of more anabolic growth factors and less proinflammatory cytokines, but the biological effect on tendons is diminished metabolism as indicated by a decrease in the synthesis of both COL1A1 and COL3A1. Together, this information suggests that minimizing leukocytes in PRP is more important than maximizing platelet numbers with respect to decreasing inflammation and enhancing matrix gene synthesis. Clinical Relevance: This study suggests that reducing leukocytes to minimize catabolic signaling appears to be more important than increasing platelets in an effort to maximize anabolic signaling. Further, a maximum biological threshold of benefit was demonstrated with regard to the number of platelets beyond which further increases in platelet concentration did not result in further anabolic upregulation. In vivo investigations documenting the use of platelets for the treatment of tendinopathy are justified as well as further in vitro characterization of the ideal PRP product for the treatment of tendinopathy and other musculoskeletal applications.

Risk factors associated with Cryptosporidium parvum infection in dairy cattle in southeastern New York State.

An observational analytical epidemiologic study was carried out to identify factors associated with the risk of infection with Cryptosporidium parvum in dairy herds in southeastern New York state. A random sample of 2943 cattle on 109 farms was selected from the target population. Fecal samples were collected from animals in three different age groups and examined for the presence of C. parvum using a quantitative centrifugation concentration flotation method. Data on intrinsic, preweaning, postweaning, maternity, and general management factors were collected and evaluated for their association with the risk of infection with C. parvum. Indices for each of these categories of management were developed from factors significantly associated with the risk of infection with C. parvum. Significant factors were identified using the logistic regression statistical technique. A final analysis, including the indices, age, and season, was performed to identify factors significantly associated with the risk of infection with C. parvum while simultaneously controlling for the effect of other factors. The farm effect was evaluated using a mixed effect model. Preweaning factors found to be significantly associated with a decreased risk of infection were: use of ventilation in calf rearing areas, daily addition of bedding, feeding of milk replacer, daily disposal and cleaning of bedding, and use of antibiotics. Postweaning factors such as moving of the animals after weaning, cleaning of soiled bedding, and use of antibiotics and ionophores as preventive measures were significantly associated with the decreased risk of an infection with C. parvum. Consideration of maternity management factors showed that winter housing of cows individually within 2 months of calving, use of fresh colostrum to feed calves, and having a concrete floor in the calving area were significantly associated with decreased risk of C. parvum infection. The total number of dairy cattle, total number of other species of agricultural animals on the farm, and the distance of the barn water source from the septic system were found to be significantly associated with increased risk of C. parvum infection. In the final analysis, the risk of infection with C. parvum was significantly decreased with an increased value of the maternity management index score. The general management significantly affected the risk of infection with C. parvum where the risk increased with the increase of the value of the index. The risk of infection significantly decreased with increase in the age of the animal.

Local and remote matrix responses to chondrocyte-laden collagen scaffold implantation in extensive articular cartilage defects.

Chondrocyte-laden collagen scaffolds were evaluated in extensive cartilage defects in an equine model. Arthroscopic techniques were used to implant a chondrocyte-collagen culture product in 15-mm defects in the lateral trochlear ridge of the femoropatellar joint of 12 horses. Ungrafted control defects were formed in the opposite joint. Groups of six horses were terminated at 4 and 8 months after implantation and the repair sites, adjacent cartilage, and remote cartilage within each femoropatellar joint examined biochemically. Eight months following surgery the relative proportions of type II collagen in grafted and ungrafted defects, determined using the ratio of cyanogen bromide cleavage products alpha 1(II)CB10/alpha 2(I)CB3,5, were not significantly different (31.57 +/- 2.76% and 26.88 +/- 2.76%, respectively). Aggrecan content was significantly improved in grafted defects (85.61 +/- 6.51 and 74.91 +/- 10.31 micrograms/mg dry weight). Cartilage surrounding grafted defects also showed improved maintenance of cartilage glycosaminoglycan content. Thus, chondrocyte grafting in collagen scaffold vehicles improved the aggrecan content in extensive cartilage defects and surrounding normal cartilage. However, given the continued disparity between repair tissue and normal cartilage aggrecan content, and the low proportion of type II collagen in grafted defects, the utility of collagen scaffolds for chondrocyte grafting of large cartilage defects seems limited.

Accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography.

The length of canine gestation is 65 days from the luteinizing hormone (LH) surge. Early and accurate determination of canine gestational age is useful for predicting and managing parturition. We performed a retrospective study on fetal measurements obtained by transabdominal ultrasonographic examination of 83 bitches (32 breeds) to estimate gestational age. Gestational age was estimated using two published tables correlating either (1). embryonic vesicle diameter (EVD), crown-rump length (CRL), body diameter (BD), and biparietal diameter (HD) to the LH surge in mid-gestational beagles or (2). BD and HD to parturition in late-gestation retrievers. Parturition date was predicted by obtaining the difference between the gestational age estimate and 65 days. Bitches were divided into four body weight (BW) groups based on nonpregnant body weight: small (<or=9 kg), medium (>9-20 kg), large (>20-40 kg), and giant (>40 kg). Mean+/-S.D. litter size (LS) was calculated for each BW group. The BW groups were then divided into small, average, or large LS groups. The accuracy of the prediction was not affected by LS but was affected by maternal body weight for small and giant BW groups only. When adjusted for weight, the accuracy of prediction within +/-1 day and +/-2 day intervals was 75 and 87%, respectively. Using stepwise logisitic regression, the most accurate prediction of parturition date was obtained when fetuses were measured at 30 days after the LH surge, regardless of body weight or LS. Parturition date predictions made after 39 days of gestation using only biparietal and BD fetal measurements were <50% accurate within +/-2 days.

Accuracy of canine parturition date prediction from the initial rise in preovulatory progesterone concentration

Accurate prediction of parturition date is useful for clinical management of canine parturition. For nearly all normal canine pregnancies, parturition occurs 64-66 days from the LH peak, the timing of which cannot be differentiated from the initial sharp rise in serum progesterone (P4) concentrations. We sought to determine by retrospective analysis if prebreeding serum progesterone concentrations could accurately predict parturition date. Serum progesterone concentrations recorded as serial samples from 63 bitches (19 breeds) were analyzed. Progesterone concentrations were measured by radioimmunoassay (RIA) or chemiluminescent immunoassay (CLIA). The CLIA method was validated for use in determining P4 concentrations in canine serum and results were comparable to those obtained with RIA. Bitches were grouped by nonpregnant body weight (BW) and litter size (LS). Day 0 (D0), the day of preovulatory rise in serum P4, was defined as the day that P4 concentration rose to > or =l.5 ng/ml and was at least twice the baseline concentration. The predicted parturition date, 65 days following the day of preovulatory rise in serum P4 (D65), was compared to actual parturition date, the day the first pup was delivered. We determined that mean P4 concentration at D0 for all BW groups was 2.02+/-0.18 ng/ml and there was significant variation in P4 concentrations between BW groups after D1. In addition, we determined that the accuracy of parturition date prediction within a +/-1, +/-2, and +/-3 day interval using prebreeding serum progesterone concentrations was 67, 90, and 100%, respectively, and that the accuracy was not affected by body weight or litter size.

Measurement of plasma cardiac troponin I concentration by use of a point-of-care analyzer in clinically normal horses and horses with experimentally induced cardiac disease

OBJECTIVE To compare cardiac troponin I (cTnI) concentrations determined by use of a point-of-care analyzer with values determined by use of a bench-top immunoassay in plasma samples obtained from clinically normal horses with and without experimentally induced cardiac disease, and to establish a reference range for plasma equine cTnI concentration determined by use of the point-of-care analyzer. ANIMALS 83 clinically normal horses, 6 of which were administered monensin to induce cardiac disease. PROCEDURES A blood sample was collected from each of the 83 clinically normal horses to provide plasma for analysis by use of the point-of-care analyzer; some of the same samples were also analyzed by use of the immunoassay. All 83 samples were used to establish an analyzer-specific reference range for plasma cTnI concentration in clinically normal horses. In 6 horses, blood samples were also collected at various time points after administration of a single dose of monensin (1.0 to 1.5 mg/kg) via nasogastric intubation; plasma cTnI concentration in those samples was assessed by use of both methods. RESULTS The analyzer-specific reference range for plasma cTnI concentration in clinically normal horses was 0.0 to 0.06 ng/mL. Following monensin treatment in 5 horses, increases in plasma cTnI concentration determined by use of the 2 methods were highly correlated (Pearson correlation, 0.83). Peak analyzer-determined plasma cTnI concentrations in monensin-treated horses ranged from 0.08 to 3.68 ng/mL. CONCLUSIONS AND CLINICAL RELEVANCE In horses with and without experimentally induced cardiac disease, the point-of-care analyzer and bench-top immunoassay provided similar values of plasma cTnI concentration.