Effectiveness of conventional and hydrosurgical debridement methods in reducing Staphylococcus aureus inoculation of equine muscle in vitro.

REASONS FOR PERFORMING STUDY: The success of primary healing of equine traumatic wounds is dependent on thorough debridement. A specific hydrosurgical debridement device (Versajet(TM) )(a) is gentle to viable tissues, yet effectively removes macroscopic contaminants and debris. We wished to investigate whether it is effective in reducing bacterial burden and whether it differs from traditional methods. No previous reports compare hydrosurgical debridement and conventional wound debridement with regard to bacterial reduction from in vitro inoculated soft tissue. OBJECTIVES: To assess the effectiveness of hydrosurgical debridement in reducing the Staphylococcus aureus load from in vitro inoculated equine muscle compared with conventional wound debridement methods. STUDY DESIGN: In vitro experimental study. METHODS: The surface of equine masseter muscle was inoculated with a S. aureus broth and subsequently debrided using one of the following 4 methods: saline irrigation; sharp debridement; saline irrigation and sharp debridement; or hydrosurgical debridement. Tissue samples for quantitative cultures were collected before and after debridement, and the colony-forming units per gram of tissue were calculated and log transformed. The reductions in bacterial counts were analysed statistically using Wilcoxon signed-rank tests and Friedman two-way ANOVA. RESULTS: Hydrosurgical debridement was more effective than conventional debridement methods in reducing the S. aureus load (P<0.05). Hydrosurgical debridement reduced the bacterial load by 99.7%, in comparison to saline irrigation and sharp debridement (87.4%), sharp debridement (82.2%) and saline irrigation (46.0%). CONCLUSIONS: Hydrosurgical debridement reduces the S. aureus load from in vitro contaminated equine muscle significantly more than conventional debridement methods.

The modified Meek technique as a novel method for skin grafting in horses: evaluation of acceptance, wound contraction and closure in chronic wounds.

REASONS FOR PERFORMING STUDY: The acceptance of skin grafts in horses is unpredictable and the final cosmetic result can be disappointing. Besides movement and infection, graft failure is often caused by chronic inflammation, inherently present during second intention healing of limb wounds in horses. In human burns affected by infection and inflammation, the acceptance of the island skin grafts of the modified Meek technique appeared to be better than meshed sheet skin grafts. HYPOTHESIS: The percentage take of Meek micrografts is higher than of other techniques; and rates of both wound contraction and epithelialisation are increased. METHODS: Large traumatic limb wounds of 13 horses healing by second intention were grafted using the modified Meek technique. Photographs of the wounds were taken at set intervals. Wound areas, and areas of acceptance and rejection were determined using a digital image post processor (Scion Image). The percentages of take, wound contraction and epithelialisation were calculated. RESULTS: The initial mean wound area was 7500 mm2. Graft acceptance was mean +/- s.d. 93.7 +/- 5.9%. Wound closure was due to contraction (55.2 +/- 11.1%) and epithelialisation (44.8 +/- 11.1%) and resulted in a 96.7 +/- 3.6% reduction of the initial wound area 29.1 +/- 6 days after grafting. All wounds showed functional and cosmetic healing. CONCLUSIONS: The method for skin grafting in horses achieved higher percentages of take than reported previously and consistent cosmetic and functional results. The grafts increased not only the rate of epithelialisation but also had a strong positive effect on wound contraction, resulting in rapid closure and smaller scars. POTENTIAL RELEVANCE: The modified Meek technique proved to be a novel technique for skin grafting equine wounds in clinical practice, which can be performed easily. The molecular background of the increase of wound contraction by the grafts may provide a clue in the search for medicinal stimulation of wound contraction during second intention healing.

Differences in polymorphonucleocyte function and local inflammatory response between horses and ponies.

REASONS FOR PERFORMING STUDY: Wound healing proceeds faster in ponies than in horses and complications during healing, such as wound infection, occur less frequently in ponies. Earlier studies suggested that this difference might be related to differences in the initial post traumatic inflammatory response. HYPOTHESIS: That polymorphonuclear leucocyte (PMN) function and profiles of humoral factors in local inflammatory processes are different in horses and ponies. METHODS: PMNs were isolated from venous blood of horses and ponies. Chemotaxis and reactive oxygen species (ROS) production was determined. Tissue cages were implanted in limbs and necks of horses and ponies and injected with carrageenan and, 3 weeks later, with LPS. In sequential samples of inflammatory exudate, the numbers of macrophages and PMNs and the production of PGE2, TNFalpha, IL-1, IL-6 and chemoattractants were determined. RESULTS: In vitro ROS production of PMNs was significantly higher in ponies than in horses, whereas in vitro PMN chemotaxis was significantly lower in ponies. In the tissue cages for both stimuli, the production of IL-1 and chemoattractants was significantly higher in ponies than in horses and remained so towards the end of the observation period in ponies. CONCLUSIONS: This study demonstrated a higher production of various inflammatory mediators by pony leucocytes. Despite the lower in vitro chemotaxis of pony PMNs, this higher in vivo production resulted in a stronger initial inflammatory response in ponies, as has been reported in studies on wound healing, through the attraction of leucocytes and triggering of the production of other cytokines. A stronger initial inflammation may promote healing by more rapid elemination of contaminants and earlier transition to repair. POTENTIAL RELEVANCE: Modulation of the initial inflammatory response might therefore be a valid option for therapeutic intervention in cases of problematic wound healing. Further, the intraspecies differences in leucocyte function may have an impact on many fields in equine medicine.

Retrospective study of primary intention healing and sequestrum formation in horses compared to ponies under clinical circumstances.

In accidental wounds, trauma and infection can result in dehiscence of primarily closed wounds and in sequestrum formation when cortical bone is exposed. In experimental studies, it has been shown that second intention healing is faster and occurs with less complications in ponies than in horses. Also, a greater initial inflammatory response was seen in ponies. Based on these experimental data, it was hypothesised that accidental wounds in ponies would heal with a lower incidence of wound dehiscence and/or sequestrum formation compared to horses. A retrospective study of 89 ponies and 422 horses with traumatic wounds was performed. The animals, wounds and treatments were categorised and related to the success rate of primary closure and to the incidence of sequestrum formation. The ponies and horses were of similar age and sex. The wounds that were treated were comparable for localisation, duration, degree of contamination and depth in both groups of animals, but there were significantly more cases with ruptured extensor tendons in ponies. Antibiotics and NSAIDs were administered significantly less often to ponies. The success rate of primary closure was significantly higher in ponies than in horses, and sequestra were formed significantly less often in ponies. It was concluded that the results of healing were better in ponies although the external conditions were less favourable. This may be associated with the differences in the initial inflammatory response after injury as found in earlier experimental work, which may result in a better local defence against wound infection.

Differences in wound contraction between horses and ponies: the in vitro contraction capacity of fibroblasts.

The contribution of wound contraction to wound closure determines the speed of second intention wound healing and it has been shown that significant differences exist with regard to both contraction and inflammatory response between horses and ponies and between various areas of the body. In this study, the contraction capacity of fibroblasts from limbs and buttocks of 4 Dutch Warmblood horses and 4 Shetland ponies was studied in vitro, in order to determine whether differences in wound contraction are due to differences in the inherent contraction capacity of the fibroblasts or to differences in tissue environmental factors, such as the inflammatory response. Fibroblasts were harvested from subcutaneous tissue, cultured and then suspended in both floating and anchored collagen gels. Contraction capacity was assessed by measuring the decrease in area of the floating gels and by measuring the microforces generated in the anchored gels using a custom-built measuring device. In the floating gels, no difference existed in the contraction capacity of fibroblasts from horses and ponies, or from limbs and buttocks. In the anchored gels, no differences existed between horse and pony fibroblasts, but the fibroblasts from the limbs started to contract significantly sooner and produced significantly higher forces than those from the buttocks. It is concluded that the in vivo differences in wound contraction between horses and ponies and between different sites of the body are not caused by differences in the inherent contraction capacity of fibroblasts. The in vitro differences between fibroblasts from limbs and buttocks are thought to be due to the lower proliferation rate and the longer culture time of the fibroblasts originating from the limbs, because mature fibroblasts can develop higher contraction forces than immature fibroblasts. This means that tissue environmental factors, such as cytokine profiles during the inflammatory response, determine the extent of contraction during wound healing. Further research should be directed towards the role of the inflammatory response in wound healing.

The effectiveness of the haemodialysate Solcoseryl for second-intention wound healing in horses and ponies.

Second-intention healing of limb wounds in horses is often problematic. Solcoseryl is a protein-free, standardized dialysate/ultrafiltrate (HD) derived from calf blood, which has been shown to improve healing in both animals and humans. The efficacy of HD in the healing of deep wounds in horses and ponies was investigated. Deep wounds of 20 by 35 mm were created on both metatarsi (skin, subcutis, periosteum) and on both femoral biceps muscles (skin, subcutis, muscle) of five horses and five ponies. The wounds on one side were treated with HD, four times a week during the period that the wounds were bandaged and once daily thereafter. The wounds on the other side were left untreated. In the first 4 weeks of the healing period HD stimulated healing but inhibited healing thereafter. This pattern was significant for all wound groups (P < 0.001). Because of this change in effect, the overall effect on wound healing over the entire period was not significant (P = 0.77). HD stimulated healing initially by provoking a greater initial inflammatory response, faster contraction and faster formation of granulation tissue. Subsequently, HD inhibited healing because it significantly delayed epithelialization and caused protracted inflammation. The effects of HD were most pronounced in the horses. Because this study distinguished between contraction and epithelialization, it could be shown that HD stimulated contraction but inhibited epithelialization. Therefore, HD is useful in horses for the treatment of deep wounds during the initial phase of healing by second intention, i.e. during the first weeks when wound contraction can be expected. Treatment should be ceased when epithelialization becomes predominant.

Differences in second-intention wound healing between horses and ponies: macroscopic aspects.

Second-intention healing of deep wounds was studied in 5 horses and 5 ponies. Standardised wounds were created on the distal limbs and hind quarters. Wounds on the metatarsi extended onto the metatarsal bone; the depth of the wounds in the femoral biceps muscle was 18 mm. The wound margins were marked by tattoos. Photographs were taken at weekly intervals to determine the wound area. The relative contribution of contraction and epithelialisation to wound closure was quantified by means of the tattoos. Swelling of the limbs was measured; and regularity and aspect of the granulation tissue were semi-quantitatively scored. Second-intention wound healing occurred significantly faster in ponies than in horses, and muscle wounds healed significantly faster than metatarsal wounds. These marked differences reflected the greater contribution of contraction to wound healing. Moreover, demarcation was seen earlier and a healthy granulation bed developed more rapidly in ponies, whereas in horses the granulation tissue remained irregular and purulent for longer. Healing of the metatarsal wounds of horses differed markedly from that of all other wounds: these wounds increased to almost twice their original size in the first 2 weeks, exuberant granulation tissue was persistent, epithelialisation started later, and contraction played a minor role in wound closure. Limb swelling was greater in horses than in ponies. Periosteal new bone formation was more extensive, and was active over a longer period in the metatarsal bones of horses than of ponies. From this study it is concluded that second-intention healing of deep wounds occurs faster in ponies than in horses. This difference can be largely attributed to a more pronounced and faster wound contraction in ponies than in horses. Therefore, attempts to improve second-intention wound healing in clinical practice should be directed at stimulation of wound contraction.

Differences in second-intention wound healing between horses and ponies: histological aspects.

The histological aspects of second-intention healing were studied in 5 horses and 5 ponies. Biopsies were taken weekly from standardised wounds on the metatarsus and femoral biceps muscle of one horse and one pony. Sections were stained to enable cell counting and the detection of DNA synthesis, fibrin, smooth muscle actin (SMA), collagen, and bacteria. In the ponies, the number of polymorphonuclear leucocytes (PMNs) was high during the first 3 weeks and subsequently decreased rapidly. In the horses, the initial number of PMNs was lower, but remained persistently elevated during the evaluation period. PMNs were found mainly in the superficial zones. Significantly more fibrin was present in the wounds of the horses. No significant differences were observed in the number of fibroblasts, the amounts of SMA and collagen. However, myofibroblasts were significantly less regularly organised in the wounds of the horses, particularly in the metatarsal wounds. The mitotic activity of the epithelium was temporally reduced in week 3. The mitotic activity of the granulation tissue was initially high but declined rapidly from week 1 onwards, with the exception of the metatarsal wounds of the horses, in which mitotic activity remained significantly higher. Histology confirmed and explained the macroscopical differences in wound healing between horses and ponies by the strict organisation of the myofibroblasts and the more effective acute inflammation in the ponies. Stimulation of the organisation of myofibroblasts and improvement of the efficacy of the inflammatory response in horses may therefore result in better second-intention wound healing in horses in clinical practice.

Long-term detection of microchimaerism in peripheral blood after pretransplantation blood transfusion.

Renal allograft survival is prolonged after pretransplantation blood transfusion. The aim of this study was to test retrospectively the development and persistence of microchimaerism after pretransplantation blood transfusion and to assess whether the type of blood transfusion (partially matched [= sharing of at least one HLA-B and one HLA-DR antigen between blood donor and recipient] versus mismatched) influences the (continued) presence of donor-type cells. A sensitive nested PCR technique based on HLA-DRB1 allele-specific amplification using sequence-specific primers (detection level: one donor cell among 10(5) recipient cells) for detection of donor cells was implemented in our laboratory. We studied 21 patients for microchimaerism in the peripheral blood compartment, following blood transfusion. Our preliminary data show that microchimaerism was detectable up to 8 weeks after blood transfusion. In all patients receiving a partially matched blood transfusion, donor-type cells were detected in the first week after transfusion, in 7/8 patients 2-4 weeks after transfusion, and in some patients up to 8 weeks after transfusion. After mismatched transfusion a tendency to shorter duration of microchimaerism was observed.

The interrelationship of calcium and magnesium absorption in idiopathic hypercalciuria and renal calcium stone disease.

PURPOSE: A decreased concentration of magnesium in the urine is a risk factor for renal calcium stone disease that may be caused by decreased enteral absorption of magnesium. We analyze the possible reciprocal influences of enteral absorption of calcium and magnesium in patients with renal stone disease. MATERIALS AND METHODS: We measured the fractional enteral absorption of 47calcium and 28magnesium in 11 patients with renal calcium stone disease, including 8 with and 3 without hypercalciuria. Two tests were performed using calcium and magnesium, respectively, followed by another test in which the enteral absorption of calcium and magnesium was measured after both cations were administered together. RESULTS: We noted no clear influence of either cation on the absorption of the other in the 3 patients without hypercalciuria. However, in the 8 hypercalciuric patients enteral calcium absorption decreased after the concurrent administration of magnesium and enteral magnesium absorption increased after the concurrent administration of calcium. Each effect was proportional to the other. CONCLUSIONS: The results of this study indicate that the oral supplementation of magnesium in patients with hyperabsorptive hypercalciuria and renal calcium stone disease is favorable because it decreases calcium absorption and increases magnesium absorption. Both factors may reduce risk factors for renal calcium stone formation.

Influence of HLA-DRB1* incompatibility on the occurrence of rejection episodes and graft survival in serologically HLA-DR-matched renal transplant combinations.

BACKGROUND: The aim of the present study was to analyze the effect of HLA-DRB1* mismatches on graft function and graft survival in 92 patients who received serologically HLA-DR split antigen-matched cadaveric renal transplants. METHODS: The polymorphic second exon of the HLA-DRB1 alleles was typed using the sequence-specific oligonucleotides technique. RESULTS: The results show that in 26 of the 92 analyzed combinations, one or more HLA-DRB1* mismatches were found (28%). The analysis of the occurrence of treatable rejection episodes during the first 3 months after transplantation demonstrated a significantly higher incidence of rejection episodes in the HLA-DRB1*-mismatched group: 18 of 26 (69%) in the HLA-DRB1*-mismatched group against 23 of 66 (35%) in the HLA-DRB1*-matched group (P(uncorr)=0.0033). However, no effect of HLA-DRB1* mismatches on graft survival was found, although in general graft survival in the whole patient group was negatively influenced by the occurrence of rejection episodes during the first 3 months after transplantation (P(uncorr)=0.0008). In contrast, in the HLA-DR4-matched donor-recipient combinations (n=28), the effect of mismatching for the HLA-DRB1*04 alleles seemed to have a pronounced effect not only on the occurrence of rejection episodes but also in the form of diminished graft survival. CONCLUSIONS: Thus, this study indicates that the existence of HLA-DRB1* allele mismatches in renal transplant recipients, matched for the serologically defined HLA-DR split antigens, is not harmful for the transplant. The exception is the HLA-DRB1*04 mismatch, which seems to be deleterious for the grafted organ.

Can low-calcium peritoneal dialysis solution safely replace the standard calcium solution in the majority of chronic peritoneal dialysis patients?

OBJECTIVE: To evaluate the use of low-calcium solution as the standard solution in chronic peritoneal dialysis patients. DESIGN: Prospective long-term follow-up study over a one-year period. SETTING: University hospital. INTERVENTIONS: The change of the calcium concentration of the dialysate from 1.75 mmol/L to 1.25 mmol/L. MAIN OUTCOME MEASURES: Serum calcium and phosphorus concentration and intact parathyroid hormone (iPTH). PATIENTS: Fifty normo- and hypercalcemic patients using the standard 1.75 mmol/L calcium solution. RESULTS: Serum ionized calcium (iCa) decreased significantly during the first six months, resulting in a significant increment of iPTH (baseline value: 0.9-79, median 9.4 pmol/L; at six months: 1.1-111, median 20.6 pmol/L; p < 0.05). In 28 patients completing the study, iPTH remained significantly elevated, despite high normal iCa. At similar changes of iCa, patients with baseline iPTH > 20 pmol/L showed a significantly higher increase in iPTH than patients with low iPTH (24.0 vs 5.0; p < 0.01), despite a more than doubled dose of alfacalcidol and calcium carbonate (mean dose of 1580 increased to 3277 mg/day). During the follow-up, 21 episodes of hypercalcemia were observed. Phosphorus control was adequate. CONCLUSIONS: Low-calcium solution cannot be used as a standard solution, especially in patients with iPTH levels indicating mild or severe hyperparathyroidism, because in these patients iPTH may rise further.

Comparison of T cell responses in patients with a long-term surviving renal allograft versus a long-term surviving liver allograft. It's a different world.

The aim of the present study was to analyze whether acquired transplantation tolerance had developed in patients with a long-term surviving renal or liver allograft. Analysis of antidonor cytotoxic T cell precursor frequencies was performed in 31 renal allograft recipients and 9 liver allograft recipients with good graft function 2 years after transplantation. The results demonstrated that, before transplantation, normal antidonor T cell responses were generated in both groups of patients. Two years after transplantation, donor-specific CTL nonresponsiveness had developed in a minority of the renal transplant recipients. In contrast, 8 out of 9 liver transplant recipients showed donor-specific mixed lymphocyte culture and CTL nonresponsiveness. These findings indicate that development of donor-specific T cell nonresponsiveness is not a common event after kidney transplantation, whereas liver transplantation seems to induce, at least in vitro, a state of donor-specific T cell nonresponsiveness.

Toxicity of OKT3 increases with dosage: a controlled study in renal transplant recipients.

In the present study we prospectively compared side effects occurring in 12 patients after the first administration of low-dose OKT3 (0.5 mg twice daily) induction therapy with those in 10 patients who were treated with a conventional dose of OKT3 (5 mg daily) for acute rejection. We also investigated cytokine release and activation of complement and neutrophils as all of these are held responsible for OKT3-induced side effects. Low-dose OKT3 resulted in a significantly decreased side effects score compared to that after a conventional dose of OKT3 (1.8 vs 5.1, p = 0.0006). Following the first administration of low-dose OKT3, TNF peak levels were significantly lower than after a conventional dose of OKT3. In contrast to our data on conventional dose OKT3 treatment, the first administration of low-dose OKT3 did not induce complement activation as reflected by C3a and C4b/c levels in plasma. Finally, the increase in neutrophil degranulation products lactoferrin and elastase-varies; is directly proportional to 1-antitrypsin was much less following 0.5 mg OKT3 than following 5 mg. We conclude that OKT3-induced toxicity is dose-dependent and is mediated not only by cytokine release but also by activation of complement and neutrophils.

Pretreatment with divided doses of steroids strongly decreases side effects of OKT3.

The aim of this study was to attenuate side effects of OKT3 by variation of the time interval between administration of corticosteroids and OKT3 in renal allograft recipients. In view of a maximal lymphocytopenia at six hours following MPNS, we postulated a greater preventive action on side effects from administration of methylprednisolone (MPNS) at six hours preceding the first dose of OKT3 compared to administration immediately before. Two groups of renal transplant patients treated for acute rejection with 5 mg OKT3 were studied. Ten patients received 500 mg MPNS six hours and ten patients one hour before administration of OKT3. We measured clinical side effects, body temperature, TNF and IL-6. There were no differences between the two groups regarding clinical side effects and peak body temperatures. However, MPNS administered six hours before administration of OKT3 diminished TNF release; MPNS one hour before decreased IL-6 release. We studied an additional group of six patients receiving 250 mg MPNS six hours before, followed by 250 mg one hour before OKT3. This group experienced significantly less side effects and lower body temperature. In addition, IL-6 levels were significantly decreased. We conclude that two times 250 mg MPNS administered six hours and one hour before the first administration of OKT3 effectively attenuates adverse reactions following administration of OKT3.

In vivo effects of IgA and IgG2a anti-CD3 isotype switch variants.

Side effects after the first administration of OKT3, a murine anti-CD3 monoclonal antibody (mAb) of the IgG2a class, are largely attributed to the release of cytokines as a result of T cell activation caused by interaction with Fc receptors (FcR) on human monocytes. As human monocytes possess FcR for murine IgG2a but not for IgA, it is expected that an anti-CD3 mAb of the IgA class causes less side-effects than an IgG2a anti-CD3 mAb of the same idiotype. To test this hypothesis we treated 20 renal transplant patients prophylactically with either IgG2a or IgA anti-CD3 mAb in a prospective randomized double-blind study. The patients received 0.5 mg anti-CD3 mAb, either IgA (T3.A) or IgG2a (T3.G2a), twice daily during 10 d. Rejection incidence after T3.A and T3.G2a was not significantly different. Side effects score after the first administration of mAb was significantly less after T3.A than after T3.G2a (0.7 vs 2.7, P = 0.002). IL-6 and gamma IFN levels increased significantly at 3 h after T3.G2a, but not after T3.A. The TNF peak level occurring at 1 h after T3.A was much lower than after T3.G2a. In plasma, complement and neutrophil activation products only increased after T3.G2a and not after T3.A. Both T3.A and T3.G2a resulted in a complete depletion of CD3+ cells, but after T3.A, CD3 depletion was of shorter duration than after IgG2a. Finally, in contrast to T3.G2a, T3.A did not affect coagulation and fibrinolysis. In conclusion, an anti-CD3 mAb of the IgA class causes hardly any cytokine release and less side-effects as compared with its IgG2a switch variant. Provided T3.A is sufficiently immunosuppressive, it is superior to OKT3.

High-affinity cytotoxic T lymphocytes after non-HLA-sharing blood transfusion--the other side of the coin.

Previously, we have shown that pretransplantation blood transfusion modulates the T cell repertoire to a great extent. Patients receiving a BT from a donor sharing one HLA haplotype with the patient (HLA-sharing BT) develop CTL nonresponsiveness against cells of the BT donor and show a selective decrease in the usage of T cell receptor V beta families. The present study has focused on the analysis of the T cell repertoire in patients receiving an HLA mismatched (non-HLA-sharing) BT. CTL precursor frequencies were measured against single class I-mismatched antigens in split-well analysis. In addition, blocking studies of CTL-target cell interaction were performed with anti-CD8 monoclonal antibodies. The results demonstrate that non-HLA-sharing BT immunizes and induces the generation of CD8 independent, high-affinity CTL against immunogenic class I-mismatched antigens. Such HLA class I antigens might become nonacceptable mismatches in subsequent organ transplantation.