Nucleic acid testing (NAT) of organ donors: is the 'best' test the right test? A consensus conference report.

Nucleic acid testing (NAT) for HIV, HBV and HCV shortens the time between infection and detection by available testing. A group of experts was selected to develop recommendations for the use of NAT in the HIV/HBV/HCV screening of potential organ donors. The rapid turnaround times needed for donor testing and the risk of death while awaiting transplantation make organ donor screening different from screening blood-or tissue donors. In donors with no identified risk factors, there is insufficient evidence to recommend routine NAT, as the benefits of NAT may not outweigh the disadvantages of NAT especially when false-positive results can lead to loss of donor organs. For donors with identified behavioral risk factors, NAT should be considered to reduce the risk of transmission and increase organ utilization. Informed consent balancing the risks of donor-derived infection against the risk of remaining on the waiting list should be obtained at the time of candidate listing and again at the time of organ offer. In conclusion, there is insufficient evidence to recommend universal prospective screening of organ donors for HIV, HCV and HBV using current NAT platforms. Further study of viral screening modalities may reduce disease transmission risk without excessive donor loss.

Pharmacological analysis of alpha(2)-adrenoceptor subtypes mediating analgesic, anti-inflammatory and gastroprotective actions.

Our previous findings suggest that alpha(2)-adrenoceptor stimulants induce gastroprotective action, the effect is likely to be mediated by alpha(2B)-adrenoceptor subtype. Clonidine (0.094 micromol/kg p.o.) and rilmenidine (0.014 micromol/kg p.o.) in gastroprotective dose range, as well as ST-91 (2.2 micromol/kg p.o.), a clonidine analogue showing higher affinity to alpha(2B)-adrenoceptor subtype than to alpha(2A)-one, inhibited the carrageenan-induced hyperalgesia in Randall-Selitto test, the antinociceptive action was reversed by yohimbine (5 micromol/kg s.c.) and the alpha(2B)-adrenoceptor antagonist prazosin (0.24 micromol/kg i.p.). Similarly, clonidine and rilmenidine in the same dose range reduced the oedema formation induced by carrageenan, yohimbine and the alpha(2A)-adrenoceptor antagonist BRL-44408 (3 micromol/kg i.p.) inhibited the anti-inflammatory effect; however, prazosin failed to affect it. These results suggest that alpha(2B/C)-like adrenoceptor subtype may be involved in the antihyperalgesic action, but not in the antiphlogistic effect of alpha(2)-adrenoceptor stimulants. The later effect may be mediated by alpha(2A)-like adrenoceptor subtype.

Analysis of the effect of neuropeptides and cannabinoids in gastric mucosal defense initiated centrally in the rat.

Increasing number of evidence suggest that gastric mucosal protection can be induced also centrally. Several neuropeptides, such as TRH, amylin, adrenomedullin, enkephalin, beta-endorphin, nociceptin, nocistatin, ghrelin or orexin given centrally induce gastroprotection and the dorsal vagal complex and vagal nerve may play prominent role in this centrally initiated effect. Since also cannabinoid receptors are present in the dorsal vagal complex, we aimed to study whether activation of central cannabinoid receptors result in gastric mucosal defense and whether there is an interaction between cannabinoids and endogenous opioids. Gastric mucosal damage was induced by 100% ethanol in rats. The cannabinoids were given intravenously (i.v.) or intracerebroventricularly (i.c.v.), while the antagonists were given i.c.v or intracisternally (i.c.). Gastric lesions were evaluated macroscopically 60 min later. Anandamide, methanandamide and WIN55,212-2 reduced ethanol-induced mucosal lesions after both peripheral (0.28-5.6 micromol/kg, 0.7-5.6 micromol/kg and 0.05-0.2 mumol/kg i.v., respectively) and central (2.9-115 nmol/rat, 0.27-70 nmol/rat and 1.9-38 nmol/rat i.c.v., respectively) administration. The gastroprotective effect of anandamide and methanandamide given i.c.v. or i.v.was reversed by the CB(1) receptor antagonist SR141716A (2.16 nmol i.c.v.). Naloxone (27.5 nmol i.c.v.) also antagonized the effect of i.c.v. or i.v. injected anandamide and WIN55,212-2, but less affected that of methanandamide. The gastroprotective effect of anandamide was diminished also by endomorphin-2 antiserum. In conclusion it was first demonstrated that activation of central CB(1) receptors results in gastroprotective effect. The effect is mediated at least partly by endogenous opioids.

Use of perfusion parameters in predicting outcomes of machine-preserved kidneys.

Perfusion parameters (PP) and early transplant outcome data from 332 consecutive ECD type kidneys machine preserved on the Waters RM-3 apparatus were reviewed and analyzed to examine the validity of using suboptimal PPs (renal resistance of .41-.60) as a criterion for discarding kidneys. Overall discard rate was 23.5%, with 55% of these having "poor" PP as part of reason for discard. PP analysis after 4 hours on the RM-3 is presented. This encompasses 280 kidneys with renal resistance .40. The PP-related discard rate in the renal resistance .41 to .60 kidneys was 51% versus 17% in the renal resistance

An evaluation of HLA cross-reactive group matching on graft survival in deceased donor kidney recipients.

Since September 20, 1999, our organ procurement organization (OPO) serving an ethnically diverse local distribution area has allocated kidneys using a cross-reactive group (CREG)-based variance. This variance awards 7 points for 0-CREG,0-DR mismatches and 6 points for 0-A,B mismatches in addition to points given for waiting time (3) and panel-reactive antibodies (PRA) > or = 80% (3). Previously, we have shown that awarding points for 0-CREG,0-DR mismatches in kidney allocation improves the access to HLA-matched transplants for racial groups, especially for the black race. In this study, we evaluated if there are outcome benefits as well. One- and 3-year uncensored graft survival data and analyses for the influence of HLA mismatching on graft outcome in black and nonblack recipients were provided by Scientific Registry of Transplant Recipients (SRTR). Overall, 1-year graft survival was 87.4% and not significantly different for blacks (86.1%, n = 467) vs nonblacks (88.8%, n = 730); 3-year graft survival was 74.6% and significantly lower P = .0001 for blacks (68.5%, n = 480) vs nonblacks (78.4%, n = 765). No significant advantage was observed for either the black or nonblack recipients in any of the HLA-mismatched categories, including the 0-CREG,0-DR mismatch group. An HLA matching effect also was not seen when data were stratified for patients nonsensitized (PRA < or = 10%) and sensitized (PRA > 10%) at the time of transplantation, except for the improved graft survival in sensitized nonblack recipients of 0- A,B,DR-mismatched grafts. Of the patients who lost their grafts and returned to the waiting list for retransplantation, the 0-A,B,DR mismatched were the least sensitized group (6%, n = 16), and there was a trend for less sensitization in the 0-CREG,0-DR-mismatched group (33%, n = 9), compared to those with other HLA mismatches (68%, n = 137). Thus, based on 1-year and 3-year follow-up data, there are no apparent graft outcome benefits for either CREG matching or conventional HLA matching in our service area, except for sensitized nonblack recipients receiving 0-A,B,DR-mismatched grafts. Such benefits may become more apparent with longer follow-up.

Congenital nuclear cataracts and uveitis in HIV-transgenic mice.

PURPOSE: Patients infected with HIV-1 develop ocular manifestations, some due to opportunistic infections and others attributed to the virus itself. Among the latter are retinal microvasculopathy and uveitis. We have analysed the ocular phenotype in HIV-transgenic mice. METHODS: We have studied T26 transgenic mice which bear a gag-pol deleted HIV-1 genome. Transgene RNA was detected by Northern analysis. Ocular pathology was assessed by conventional histology, immunostaining for gp120 envelope protein, and in situ apoptosis detection with end-labelling. RESULTS: Abnormalities of lens epithelial cell development were detected as early as embryonic day 14.5. Histological changes included the malformation of an embryonal lens nucleus and poor closure of the lens suture lines. This resulted in congenital nuclear cataracts, as occur in congenital viral infections in human patients. In the adult animals, lenses were notable for extensive vacuolation, liquefaction, and degeneration of the cortex. Mild iridocyclitis and vitritis were also noted in adult transgenic mice. Immunostaining demonstrated the expression of gp 120 envelope protein within the lens epithelial and fibre cells. End-labelling with terminal deoxyribonucleotidyl transferase showed increased numbers of apoptotic cells in the adult lens. CONCLUSIONS: These findings suggest that one or more HIV-1 proteins are associated with congenital nuclear cataract formation and uveitis in HIV-transgenic mice.

Prolonged leaching time of peptide antibiotics from acrylic bone cement.

The leaching time of antibiotics in growth inhibitory concentrations from polyacrylic bone cements was determined using Escherichia coli, methicillin resistant Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, and Pseudomonas aeruginosa as target bacteria. The leaching time of the peptide antibiotics vancomycin and polymyxin B nonapeptide was considerably longer than that of gentamicin, novobiocin, and erythromycin. Among the nonpeptide antibiotics, the leaching time of novobiocin lasted longer than did that of gentamicin. The acylated parent polymyxin B antibiotic leached for a considerably shorter period than did the deacylated polymyxin B nonapeptide derivative, suggesting that the lipids impede the leaching process from the cement. Cement beads impregnated with polymyxin B nonapeptide and introduced into the tibia of three rabbits receiving oral novobiocin protected bone infection against a challenge of Pseudomonas aeruginosa that otherwise caused infection in tibias containing gentamicin impregnated cement beads. The peptide antibiotics alone or in combination with other antibiotics (polymyxin B nonapeptide and novobiocin) impregnated in cements for orthopaedic procedures may provide longer periods of protection against a wide range of bacterial pathogens.

Isoform specificity of commercially-available anti-TGF-beta antibodies.

The transforming growth factor-beta (TGF-beta) cytokine family has important and complex effects on many biologic processes. Mammals have three TGF-beta isoforms which differ in their primary amino acid sequence, receptor binding characteristics, distribution, and function. Characterization of TGF-beta production and localization is critically dependent upon appropriate reagents, including antibodies. We have analyzed the isoform specificity of eight commercially-available TGF-beta antibodies, including one monoclonal antibody and seven polyclonal antibodies. We carried out semi-quantitative Western blot analysis using recombinant TGF-beta1, beta2, and beta3 as targets. We found that sensitivity and isoform specificity are dependent in part upon the presence or absence of reducing conditions. The antibodies tested showed a broad range of sensitivity, with an ability to detect 50 pg to 20 ng. Cross-reactivity with another, incorrect isoform was seen with several antibodies, and ranged from 0.2% to 42%. Nevertheless, we identified TGF-beta antibodies directed against each isoform which provide moderate-to-high sensitivity and specificity when used in Western blot analysis. These results may have relevance for investigators who wish to detect particular TGF-beta isoforms with techniques other than Western blot analysis, particularly when these techniques involve denatured proteins.

Renal expression of fibrotic matrix proteins and of transforming growth factor-beta (TGF-beta) isoforms in TGF-beta transgenic mice.

Renal pathology in mice that are transgenic for the murine albumin enhancer/promoter linked to a full-length porcine transforming growth factor-beta1 (TGF-beta1) gene has been described previously. In these mice, transgene expression is limited to the liver and the plasma level of TGF-beta is increased. The earliest renal pathologic change is glomerulosclerosis, at 3 wk of age, and this is followed by tubulointerstitial fibrosis. In this study, it was hypothesized that circulating TGF-beta1 increases renal extracellular matrix accumulation and activates local TGF-beta gene expression. Immunostaining at 5 wk revealed increased amounts of collagen I and III within the mesangium, glomerular capillary loops, and interstitium, while the amount of collagen IV was normal. Similarly, Northern analysis showed increased expression of mRNA encoding collagen I and III, as well as biglycan and decorin, while the expression of collagen IV was unchanged. These changes began as early as 1 wk of age, a time before the appearance of glomerulosclerosis. To evaluate matrix degradation, collagenase IV activity was evaluated by gelatin zymography and an increase in matrix metalloproteinase-2 was found. Finally, the production of tissue inhibitors of metalloproteinase was evaluated. Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA was increased 18-fold, while TIMP-2 and TIMP-3 were unchanged. In 2-wk-old transgenic kidney, local expression of TGF-beta1, beta2, and beta3 protein was similar to wild-type mice. In 5-wk-old transgenic mice, TGF-beta1 and beta2 protein was present in increased amounts within glomeruli, and renal TGF-beta1 mRNA was increased threefold. It is concluded that elevated levels of circulating TGF-beta1 may act on the kidney to increase matrix protein production and decrease matrix remodeling. Only after glomerulosclerosis is established does local glomerular overproduction of TGF-beta become manifest.

Captopril prevents nephropathy in HIV-transgenic mice.

Transgenic mice (T26) bearing the envelope, regulatory, and accessory genes of HIV- I develop renal disease resembling human HIV-associated nephropathy (HIVAN). Effects of vehicle (VEH) and the angiotensin-converting enzyme inhibitor captopril (CAP) were examined in wild-type (WT) or T26 mice treated from 7 to 100 d of age. Mortality was lower in CAP T26 mice (30 mg/kg: 8%; 100 mg/kg: 12%) than VEH T26 mice (52%). The urinary protein/creatinine ratio was increased in VEH T26 mice (19.5+/-7.60) versus WT mice (6.1+/-0.83), but not in low-dose (7.3+/-0.94) or high-dose (8.2+/-1.02) CAP T26 mice. Blood urea nitrogen was higher in VEH T26 mice (52+/-16.2 mg/dl) than VEH WT mice (24+/-0.8). Blood urea nitrogen was also elevated in CAP WT (high dose: 43+/-2.1 mg/dl) and T26 mice (high dose: 42+/-2.4 mg/dl). Glomerular injury was higher in VEH T26 mice (6.8+/-0.58) than VEH WT mice (0.2+/-0.08) or CAP T26 mice (low dose: 1.1+/-0.17; high dose: 0.7+/-0.13). Tubulointerstitial injury was also greater in VEH T26 mice (1.1+/-0.10) than VEH WT mice (0.2+/-0.08) or CAP T26 mice (low dose: 0.4+/-0.10; high dose: 0.3+/-0.10). These data validate recent nonrandomized studies of captopril in HIV-infected patients, and suggest that an angiotensin-converting enzyme substrate is an important mediator in HIVAN. A randomized placebo-controlled trial of captopril in HIVAN may be warranted.

Transarticular tumor invasion via ligamentum teres. A clinical-pathologic study of 12 patients.

We studied 12 patients who were operated on for malignant tumors in and around the hip joint. A correlative study, including preoperative staging studies and anatomical-pathologic aspects of the hip joint, was performed. In 4 of the 12 patients, we found direct histologic evidence of tumor invasion from the head of the femur through the ligamentum teres to the acetabular fovea and vice versa. It seems that the ligamentum teres is a potential route for transarticular spread of a tumor.

Malignant bone and soft tissue tumors of the shoulder girdle. A retrospective analysis of 30 operated cases.

From 1988 to 1995, 30 patients (16 men) with malignant bone (n 23) and soft tissue (n 7) tumors of the shoulder girdle underwent surgery in our department. The mean age was 34 (6-80) years. 26 patients had primary and 4 had metastatic lesions. The average follow-up period was 3 (2-8) years, at the end of which 18 patients showed no evidence of disease, 2 were alive with disease, and 10 had died (9 because of tumor). 25 of the operations were limb-sparing procedures, while the other 5 were major amputations. Radical resection was performed in 4 patients, wide resection in 25 and marginal resection in 1. Local recurrence was observed in 2 patients. 10 patients with stage IIB tumors of the proximal humerus underwent extraarticular humeral and glenoid resection. Reconstruction was performed with either a modular or an improvised implant. Following surgery, those patients had a concave contour of the shoulder and poor abduction ability. Overall functional outcome was good in 18 patients, moderate in 11 and poor in 1. No correlation was found between functional outcome and reconstruction technique.

Prognostic factors and early resumption of cyclosporin A in renal allograft recipients with thrombotic microangiopathy and hemolytic uremic syndrome.

Biopsy-proven thrombotic microangiopathy (TMA) was found in 22 of 436 (5%) renal transplant recipients, with similar incidence in recipients of cadaver or living related allografts. All patients with TMA presented different degrees of severity of the hemolytic uremic syndrome (HUS). Prognosis was poor when HUS occurred shortly after transplant in recipients of cadaveric kidneys (55% graft loss). It was more favorable when HUS occurred later in the post-transplant course or in recipients with allografts from living related donors, irrespective of time of occurrence. Other factors such as extent of TMA, degree of thrombocytopenia, hemolysis or renal dysfunction were not predictive of graft loss. Cyclosporine was resumed in 14 of 16 recipients shortly after clinical recovery without recurrence of HUS. In conclusion, HUS carries poor prognosis when occurring shortly after transplant in cadaver kidney recipients. Once the graft function improves, cyclosporine can be safely resumed.

High dose tumor necrosis factor-alpha and melphalan administered via isolated limb perfusion for advanced limb soft tissue sarcoma results in a >90% response rate and limb preservation.

BACKGROUND: Recombinant tumor necrosis factor-alpha (rTNF-alpha) is a highly potential antineoplastic agent. However, its systemic administration in humans has resulted in a life-threatening septic shock-like syndrome, and its use has been abandoned. The administration of high dose rTNF-alpha and melphalan via isolated limb perfusion (ILP) eliminated the systemic side effects and was shown to be very effective for metastatic melanoma confined to the limb. The purpose of the current study was to assess the role of rTNF-alpha and melphalan administered via ILP in patients with soft tissue sarcoma. Amputation is unavoidable in 10% of these patients despite aggressive conventional therapy. Limb preservation was the objective in this select group of candidates for amputation or mutilating surgery. METHODS: During a 36-month period, 35 patients with high grade soft tissue sarcoma underwent 41 ILPs with high dose rTNF-alpha (3-4 mg) and melphalan (1-1.5 mg/kg). There were 21 males and 14 females. The mean age was 48 years (range, 14-80 years). Histologic subtypes included malignant fibrous histiocytoma, synovial, liposarcoma, malignant schwannoma, desmoid, clear cell, epithelioid, rhabdomyosarcoma, leiomyosarcoma, and unclassifiable. Twenty-one patients presented with recurrent and 14 with very extensive primary tumors. The tumors were located in the upper extremity in 8 patients and in the lower extremity in 27 patients. Twenty-five patients were candidates for amputation and 10 for extensive mutilating surgery. ILP was performed via the corresponding vessels proximal to the tumor. Six patients with partial response (PR) insufficient to render them resectable underwent a second ILP. With the exception of 4 of 9 patients with multifocal lesions and 1 who refused surgery, resection of the residual tumor or tumor bed or limb was performed 6-8 weeks after ILP. RESULTS: Marked tumor softening occurred within 48 hours, and in tumors protruding through the skin hemorrhagic necrosis was evident within 24 hours. The overall response rate was 91%. Thirteen patients (37%) had a complete response and 19 (54%) had a PR; in 5 of these patients > 90% necrosis of the tumor occurred. In 3 patients (8.5%), only minimal regression was observed (stabilization of disease). The rate of limb sparing was 85% (29 of 34 patients). CONCLUSIONS: The combination of high dose rTNF-alpha and melphalan given via ILP appears to be effective in patients with advanced soft tissue sarcoma confined to the limb, achieving a high response rate and limb preservation.