Expression of biologically active recombinant porcine interleukin-12 from Escherichia coli.

The control of viral infections is of critical importance to livestock industries worldwide and is highlighted by costly infection outbreaks, such as that seen with foot and mouth disease virus. To ameliorate the impact of increasing problems with viral infections, new vaccine and anti-viral strategies are required and a greater understanding of the anti-viral response is essential. Furthermore, in pigs, evidence is still being gathered on the components of a defined anti-viral immune response. However, this has been greatly improved by the recent cloning and expression of critical cytokines involved in the anti-viral response. To assess the use of recombinant porcine interleukin-12 (rPoIL-12) as an immunotherapeutic and immunomodulator of swine, we have cloned and expressed rPoIL-12 as a single-chain fusion protein from Esherichia coli (E. coli). The fusion encodes the p40 and p35 subunits, linked by a glycine-serine linker and expressed as a C-terminal 6xHis tagged protein. rPoIL-12 stimulated the proliferation of human lymphoblasts and its activity on porcine cells was demonstrated by the ability of rPoIL-12 to increase the mRNA expression of porcine interleukin-18 receptor-alpha (poIL-18Ralpha) from porcine peripheral blood mononuclear cells (PoPMBCs). This data supports the inclusion of E. coli produced rPoIL-12 in immunomodulation strategies in the pig.

Bite-force development, metabolic and circulatory response to electrical stimulation in the canine and porcine masseter muscles.

The development of fatigue was investigated by electrical stimulation in 15 domestic pigs (1 yr old, 70-90 kg body weight) and seven adult dogs (3 yr old, 45 kg body weight). After anaesthesia, silver electrodes were implanted in the anterior and posterior parts of the right masseter muscles. The contralateral muscle was used as control. The bite force was measured. Muscle biopsies were obtained from the anterior, central and posterior parts, and were immediately frozen in liquid nitrogen. A fluorometrical analysis by enzymatic methods for glycogen, glucose, creatine phosphate, NAD, NADH, lactate and pyruvate was made. Blood flow was measured by 133Xe wash-out; oxygen consumption was monitored with an oxygen electrode. The porcine masseter was continuously stimulated for 60 min (100 V, 4 Hz and 2 ms). The canine masseter was intermittently stimulated (100 V, 20 Hz and 2 ms). The contraction was repeated four times, with a 10-min rest between. The porcine masseter could sustain longer endurance times than the canine masseter, which was easily fatigued. A marked substrate depletion was evident. The precontractional contents of glycogen, glucose and creatine phosphate were reduced. Lactate accumulation was evident (2-4 times more in the porcine and 4-8 times more in the canine masseter). The NADH concentration increased and the NAD content decreased. Blood-flow impairment (80% reduction in the dog, 60% in the pig) was observed. After the contraction phase, there was a hyperaemia (58% elevation of blood flow in the pig masseter, 45% in the canine). The oxygen tension followed in magnitude and time the blood-flow changes. These circulatory variables returned to normal after recovery. The high degree of substrate depletion, blood-flow impairment and a simultaneous decrease in oxygen transport to the contracted muscle, in combination with a prominent lactate accumulation, may induce a decrease in bite-force production.

Fatigue development during electrical stimulation in the masseter muscle of rhesus monkeys (Macaca mulatta).

Fatigue development was investigated in five adult female rhesus monkeys, 9-11 yr old (mean weight, 4.6 kg). After sedation and anaesthesia, silver electrodes were implanted in the anterior and posterior parts of the right masseter; the contralateral muscle was used as a control. The bite force was monitored. Muscle biopsies were obtained from the central part of the masseter and were immediately frozen in liquid nitrogen. After freeze-drying a fluorometric analysis using enzymatic methods for measuring levels of glycogen, glucose, lactate, pyruvate, creatine phosphate, creatine, NADH and NAD was made. The masseters were stimulated for 3 min (100 V, 4 Hz and 2 ms). After a 5-min rest period the stimulation was repeated with the same voltage, frequency and duration. The rhesus monkey masseters were easy to fatigue. After the stimulations 25% of the initial bite force remained. A marked substrate depletion was evident. The precontraction values of glycogen, glucose and phosphocreatine were reduced. The NADH concentration increased and the NAD content decreased. An accumulation of waste products was observed; the pyruvate increased by 92% and the lactate increased by a factor of 3. The substantial substrate depletion in combination with a prominent metabolic waste-product accumulation may induce a decrease in bite-force production.

Gross anatomy of the mandibular joint and masticatory muscles in the domestic pig (Sus scrofa).

The mandibular joint (MJ) and masticatory muscles in 15 domestic pigs, aged 1 year, were investigated during and after dissection. The joint consisted of a fossa, disc and condyle surrounded by a ligamentous capsule. The fossa was almost flat, slightly convex antero-posteriorly and concave medio-laterally; the disc was biconcave and the condyle oval. The long axis of the condyles formed an anteriorly orientated angle to the frontal plane. The masticatory muscles were similar to human ones except for the pig zygomatico-mandibular muscle. The resemblance between the anatomy of the porcine and human joint and masticatory muscles makes the pig a useful animal for functional studies of the masticatory system.

Gross anatomy of the craniomandibular joint and masticatory muscles of the dog.

The craniomandibular joint (CMJ) and masticatory muscles in 15 dogs (Canis familiaris) were dissected. The major structures were fossa, disc and condyle surrounded by a thin fibrous capsule. The horizontally orientated temporal fossa had a pronounced tubercle anteriorly. Posteriorly, a retroarticular process curved backwards. This fossa and process closely surrounded the condyle. The disc was thin. The cylindrically-shaped condyle was elongated in the medio-lateral direction, situated at right angles to the sagittal plane. The lateral pterygoid muscle was poorly developed; the temporalis was by far the largest masticatory muscle. These findings support the assumption that the movements of the canine mandible are mainly hinge-like. Furthermore, the canine CMJ differs in many respects from the corresponding anatomical structures in the domestic pig, as well as in man.

Frequency-dependent fatigue development during electrical stimulation in the masseter muscle of pigtail monkeys.

Low-frequency fatigue was investigated in nine female and one male adult pigtail monkeys (Macaca nemestrina) with a mean weight of 5.3 kg (range 4.3 to 6.5 kg). After sedation and anesthesia, silver electrodes were inserted into the anterior and posterior parts of the right masseter muscle. The contralateral muscle was used as a control. The masseter muscles were stimulated for 3 minutes (4 Hz, 2 ms, 100 V). After a 5-minute rest period, the stimulation was repeated with the same duration and voltage but at a higher frequency of 8 Hz. Bite forces were measured, and muscle biopsies were obtained from the central part of the right masseter and immediately frozen in liquid nitrogen. After freeze-drying, a fluorometric analysis that used enzymatic methods for measuring levels of glycogen, glucose, lactate, pyruvate, creatine phosphate, nicotinamide-adenine dinucleotide (NAD), and reduced NAD (NADH) was performed. The bite force decreased by 12% after the initial 3 minutes of work. After the second contraction the bite force decreased to 56%. Prominent substrate depletion was observed. The precontraction levels of glycogen, glucose, and phosphocreatine were all reduced. The NADH and the NAD concentrations increased. An accumulation of metabolites was evident. The pyruvate increased by 32% and lactate levels increased by a factor of 3. The male measurements were comparable to the nine female measures for each assessment. The substantial substrate depletion in combination with a prominent accumulation of metabolites may contribute to the development of low-frequency fatigue.

Controversies on anatomy and function of the ligaments associated with the temporomandibular joint: a literature survey.

Varying opinions are found in the literature regarding the role of the ligaments associated with the temporomandibular joint, eg, with respect to recording of mandibular positions and diagnosis and to treatment of patients with temporomandibular disorders. A literature survey was done to explore controversies of anatomy and function of the temporomandibular joint ligaments. Only 20 research-based anatomic/functional articles were registered in the Index Medicus system under the selected headings from 1897 to 1995. By examining the reference lists of the articles found, 22 more articles not included in Index Medicus were identified. Thus, the total number of original publications concerning the temporomandibular ligaments was limited. Although the conventional textbook descriptions of the anatomy of the ligaments are fairly consistent, there are several controversies and unanswered questions. Information about the functional role of the ligaments is especially insufficient. The results of this survey indicate that there is a need for additional anatomic and functional investigations of ligaments associated with the temporomandibular joint.

Use of buccal cells collected in mouthwash as a source of DNA for clinical testing.

CONTEXT: To maximize the participation rate in population genetic studies, alternatives to invasive whole blood collection are increasing. One such alternative is buccal epithelial cell collection, which, in contrast to venipuncture and finger sticks, is painless. Buccal cells, if collected and purified efficiently, offer an acceptable source for DNA to be used in research and clinical applications. OBJECTIVE: To develop a noninvasive sampling method for collecting cells for routine DNA testing in a clinical laboratory setting. DESIGN: Five factors were used to evaluate several brands of mouthwash: (1) compatibility with the DNA purification chemistry, (2) DNA yield, (3) DNA quality, (4) DNA stability at room temperature, and (5) mouthwash taste. Next, an optimization study was undertaken to maximize DNA yield. Finally, a validation study was undertaken with the optimized protocol to test a panel of 14 donors for DNA yield and performance and to test for the stability of DNA held in mouthwash. SETTING: Industrial research and development laboratory. RESULTS: Of 5 mouthwashes tested, Scope brand mouthwash received the highest overall ranking. The addition of proteinase K and glycogen to the protocol significantly enhanced DNA yields, with a test panel (n = 14) giving a range of 12 to 60 microg of DNA per donor. In a 4-week room temperature stability study, the DNA in mouthwash samples was found to be stable for at least 2 weeks. CONCLUSION: A clinically validated DNA purification chemistry was adapted to a noninvasive specimen collection method. This method used a commercially available mouthwash, Scope, to collect buccal epithelial cells for the preparation of high-quality DNA in high yield.

Masseter muscle performance. Significance of structure and metabolism. A morphological and experimental study.

The aim of this thesis was to characterize structural factors in masticatory systems relevant to functional evaluations and to elucidate the effect on energy metabolism of electrically induced jaw muscle fatigue. An omnivorous masticatory system (the domestic pig) was compared morphologically with a carnivorous (the dog). Porcine masseter muscles were evaluated by ATP-ase histochemistry as well as with NADH-dehydrogenase and PAS-staining. Contractional characteristics were obtained from the porcine and canine masseters by electrical stimulation. The 133Xenon clearance technique and a flexible oxygen electrode were employed. A bite-force transducer was used. The porcine craniomandibular joint (CMJ) lacked a pronounced mandibular fossa and had anteriorly orientated cylindrical condyles. The dog CMJ comprised a cylindrical condyle orientated at right angles to the satittal plane and medially inclined. The pronounced mandibular fossa and marked tubercle, together with a well-developed retro-articular process, surrounded the condyle. The masticatory muscles were the same in the two species, except for the pig's zygomatico-mandibular muscle. The ATP-ase technique failed to reveal type II:B-fibres in the porcine masseter after acid and alkaline preincubation and it was not possible to separate fibre types by glycogen-staining and NADH-dehydrogenase histochemistry. These findings diverged from the pig soleus histochemical profile (type II:B-fibres 60%). The quantitative evaluation revealed 75% type II:A-fibres in the porcine masseter. No statistically significant difference was found between the various fibre-type diameters in the porcine masseter. The mean fibre Type diameter was larger in the porcine masseter than in the soleus muscle. Type II-fibres were more frequently found on the edge of the fascicles. The bite force recordings showed that the porcine masseter was capable of long endurance performance, in contrast to the easily fatigued canine masseter. Significant reductions of intramuscular substrates and a considerable lactate accumulation were observed. The NADH/NAD-shuttle was oppositely directed in the two species. The blood-flow recordings revealed a marked blood-flow impairment during contraction, followed by a prominent post-exercise hyperaemia. The pO2 recordings were closely related in time as well as in mangnitude to the blood flow. It is thus concluded, based on morphological observations, that the porcine masticatory system bears resemblance to the human situation. In the canine masseter muscle, a relationship was found between metabolism and mechanical bite-force output. This correlation was not so evident in the porcine masseter. Induced jaw muscle hyperactivity may lead to a reduced energy and redox state and, as a late consequence, to fatigue.

Anatomy of the moose craniomandibular joint (Alces alces, Linnaeus, 1758).

the craniomandibular joints (CMJ) were investigated in 10 Swedish mooses (eight cows: five 1-year-old, two two-year-old, and one 7-year-old) together with one bull moose (2 years) and a yearling. The CMJs, situated at a high distance from the occlusal plane of the tooth rows, comprised a temporal component, a disc and a condyle. The temporal component lacked a mandibular fossa as well as articular eminence. The joint surface was oval, almost flat and convex. The condyle had about two-thirds of its length medio-laterally, cylindrical in shape and from a posterior view medially inclined. The articulating surface was divided into a horizontal part located on the superio-anterior condyle, and a vertical part medially situated. The disc was oval in shape and revealed two synovial chambers totally surrounded by a soft tissue lining.

Microanatomy of the moose temporomandibular joint (Alces alces; Linnaeus, 1758).

Temporomandibular joint (TMJ) histology was investigated in 8 female Scandinavian moose. 5 were 1-year-old with a carcass weight (cw) of 125-140 kg, and 3 were 2-years-old (160-175 kg cw). The condylar articular surface consisted of a connective tissue lining with parallel collagen fibres. Numerous blood vessels were observed adjacent to the joint chamber. Below the fibrous layer, a proliferative cellular zone of undifferentiated mesenchymal cells was situated. These cells differentiated into chondroblasts and hypertrophied chondrocytes. Further down, an endochondral ossification process was initiated. Vertically directed invaginations were observed. A similar cellular organization was identified in the temporal component. However, the undifferentiated mesenchymal cell layer was discontinuous. The disc showed dense collagen bundles without main alignment. Vessels were identified throughout the entire disc. The results indicate that the cellular organization of the moose TMJ is similar to the TMJ histology found in other mammals but differences do occur.

Blood-flow and metabolic response in a contracting canine masticatory muscle.

The mechanical and metabolic characteristics of the canine masseter muscle of seven adult dogs, aged 3 years, weighing 45-55 kg, were evaluated by electrically provoked contractions. The masseter muscle was exposed by careful surgery and stimulated by two silver electrodes placed in the anterior and posterior aspects of the muscle. The bite force was measured with a force transducer. Glycogen, glucose-contents together with lactate, pyruvate, creatine phosphate, NADH and NAD were analysed fluorimetrically. Intra-muscular oxygen tension was continuously recorded by a flexible silver electrode. Blood-flow was measured by the 133Xenon clearance technique. Biopsies were taken at rest and after stimulation. The registered bite force revealed an initial peak, representing maximal force production and thereafter a rapid decline in power output. During the following stimulation events the bite force curve showed decrease in amplitude concerning maximal force as well as duration of bite force production. The blood-flow and oxygen tension were significantly reduced at the end of stimulation. However, after recovery these parameters were normalised to initial values. The glycogen and glucose were depleted after stimulation. The lactate concentration increased about three to four times. The observed changes in NAD and NADH indicate a hypoxic situation in the muscle cells. The oxygen deficit in conjunction with the intramuscular blood-flow impairment and the metabolite variations reflect the energy and redox state in the muscle and may contribute to the decline in bite force production.