Association of single nucleotide polymorphisms in pro-inflammatory cytokine and toll-like receptor genes with pediatric hematogenous osteomyelitis.

Hematogenous osteomyelitis (HO) is a bone infection wherein bacteria penetrate to the bone through the blood stream. Several single nucleotide polymorphisms (SNPs) have been associated with susceptibility to infectious diseases. In this study, we investigated the contribution of SNPs in interleukin (IL)-1B1 (rs16944), IL1A (rs1800587), IL1B (rs1143634), toll-like receptor (TLR)-2 (rs3804099), TLR4 (rs4986790), TLR4 (rs4986791), IL1R (rs2234650), tumor necrosis factor (TNF)-α (rs1800629), TNF (rs361525), and IL1RN (rs315952) towards the development of HO in Saudi patients and compared to healthy controls. Fifty-two patients diagnosed with HO and 103 healthy individuals were genotyped. The frequencies of genotypes GG (rs16944) and AA (rs16944) were lower and higher in patients [odds ratio (OR) = 0.34, Pc = 0.05] and controls (OR = 1.33, Pc = 0.05), respectively, suggesting that SNPs at this locus could alter HO susceptibility. In addition, the patients and controls exhibited lower and higher frequencies of the alleles G (rs16944) (OR = 0.43, Pc = 0.007) and A (rs16944) (OR = 2.32, Pc = 0.007), respectively. The expression of alleles C (rs3804099) and T (rs3804099) were higher in patients (OR = 2.05, Pc = 0.04) and controls (OR = 0.49, Pc = 0.04), respectively. In conclusion, SNPs at rs16944 and rs3804099 were found to be associated with HO in the Saudi population.

Post-traumatic immunosuppression is reversed by anti-coagulated salvaged blood transfusion: deductions from studying immune status after knee arthroplasty.

Major trauma increases vulnerability to systemic infections due to poorly defined immunosuppressive mechanisms. It confers no evolutionary advantage. Our objective was to develop better biomarkers of post-traumatic immunosuppression (PTI) and to extend our observation that PTI was reversed by anti-coagulated salvaged blood transfusion, in the knowledge that others have shown that non-anti-coagulated (fibrinolysed) salvaged blood was immunosuppressive. A prospective non-randomized cohort study of patients undergoing primary total knee arthroplasty included 25 who received salvaged blood transfusions collected post-operatively into acid-citrate-dextrose anti-coagulant (ASBT cohort), and 18 non-transfused patients (NSBT cohort). Biomarkers of sterile trauma included haematological values, damage-associated molecular patterns (DAMPs), cytokines and chemokines. Salvaged blood was analysed within 1 and 6 h after commencing collection. Biomarkers were expressed as fold-changes over preoperative values. Certain biomarkers of sterile trauma were common to all 43 patients, including supranormal levels of: interleukin (IL)-6, IL-1-receptor-antagonist, IL-8, heat shock protein-70 and calgranulin-S100-A8/9. Other proinflammatory biomarkers which were subnormal in NSBT became supranormal in ASBT patients, including IL-1ß, IL-2, IL-17A, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and annexin-A2. Furthermore, ASBT exhibited subnormal levels of anti-inflammatory biomarkers: IL-4, IL-5, IL-10 and IL-13. Salvaged blood analyses revealed sustained high levels of IL-9, IL-10 and certain DAMPs, including calgranulin-S100-A8/9, alpha-defensin and heat shock proteins 27, 60 and 70. Active synthesis during salvaged blood collection yielded increasingly elevated levels of annexin-A2, IL-1ß, Il-1-receptor-antagonist, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IFN-γ, TNF-α, transforming growth factor (TGF)-ß1, monocyte chemotactic protein-1 and macrophage inflammatory protein-1α. Elevated levels of high-mobility group-box protein-1 decreased. In conclusion, we demonstrated that anti-coagulated salvaged blood reversed PTI, and was attributed to immune stimulants generated during salvaged blood collection.

Anti-self HLA may be clonally expressed.

A monolayer absorption technique was used to test the hypothesis that killer cells directed to self HLA-associated minor histocompatibility antigens (H-Y) were divisible into subsets. The results showed that sensitized killer cells, which recognized two combined antigens HLA-A2; H-Y and HLA-B7; H-Y could indeed be divided into two populations. One was directed to HLA-A2; H-Y and the other to HLA-B7; H-Y. These results can be interpreted in the context of the altered self hypothesis. However, when interpreted in the context of the dual recognition hypothesis, they strongly suggest that independant clones of killer T cells exist which are committed to the recognition of self HLA.

Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings.

PURPOSE: To compare outcomes of bone marrow transplants for leukemia from HLA-identical siblings, haploidentical HLA-mismatched relatives, and HLA-matched and mismatched unrelated donors. PATIENTS: A total of 2,055 recipients of allogeneic bone marrow transplants for chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) were entered onto the study. Transplants were performed between 1985 and 1991 and reported to the International Bone Marrow Transplant Registry (IBMTR). Donors were HLA-identical siblings (n = 1,224); haploidentical relatives mismatched for one (n = 238) or two (n = 102) HLA-A, -B, or -DR antigens; or unrelated persons who were HLA-matched (n = 383) or mismatched for one HLA-A, -B, or -DR antigen (n = 108). HLA typing was performed using serologic techniques. RESULTS: Transplant-related mortality was significantly higher after alternative donor transplants than after HLA-identical sibling transplants. Among patients with early leukemia (CML in chronic phase or acute leukemia in first remission), 3-year transplant-related mortality (+/-SE) was 21% +/- 2% after HLA-identical sibling transplants and greater than 50% after all types of alternative donor transplants studied. Among patients with early leukemia, relative risks of treatment failure (inverse of leukemia-free survival), using HLA-identical sibling transplants as the reference group, were 2.43 (P < .0001) with 1-HLA-antigen-mismatched related donors, 3.79 (P < .0001) with 2-HLA-antigen-mismatched related donors, 2.11 (P < .0001) with HLA-matched unrelated donors, and 3.33 (P < .0001) with 1-HLA-antigen-mismatched unrelated donors. For patients with more advanced leukemia, differences in treatment failure were less striking: 1-HLA-antigen-mismatched relatives, 1.22 (P = not significant [NS]); 2-HLA-antigen-mismatched relatives, 1.81 (P < .0001); HLA-matched unrelated donors, 1.39 (P = .002); and 1-HLA-antigen-mismatched unrelated donors, 1.63 (P = .002). CONCLUSION: Although transplants from alternative donors are effective in some patients with leukemia, treatment failure is higher than after HLA-identical sibling transplants. Outcome depends on leukemia state, donor-recipient relationship, and degree of HLA matching. In early leukemia, alternative donor transplants have a more than twofold increased risk of treatment failure compared with HLA-identical sibling transplants. This difference is less in advanced leukemia.

Allogenotypes defined by short DQ alpha and DQ beta cDNA probes correlate with and define splits of HLA-DQ serological specificities.

HLA-DR and -DQ serotyped cell lines and peripheral blood leucocytes were analysed by Southern blot allogenotyping. Using a short DQ beta cDNA probe, a DQ beta allelic series was defined by restriction fragment length polymorphism (RFLP) with the restriction endonuclease TaqI. This DQ beta allelic series correlates with, and defines splits of, the HLA-DQ serological specificities DQw1 (DQ beta 1a and DQ beta 1b RFLPs), DQw2 (DQ beta 2a and DQ beta 2b RFLPs) and DQw3 (DQ beta 3a and DQ beta 3b RFLPs). By sequential use of a short DQ alpha cDNA probe a second, DQ alpha allelic series is defined by RFLP. This series correlates to a lesser extent than DQ beta RFLPs with the HLA-DQ serological specificities. Thus, two DQ alpha RFLPs correlate with a single DQ serotype (DQ alpha 1a and DQ alpha 1c with DQw1), but three DQ alpha RFLPs correlate with more than one DQ serotype (DQ alpha 1b with DQw1 and DQw3; DQ alpha 2 with DQw2 and DQw3; DQ alpha 3 with DQw2 and DQw3). Individual DQ beta and DQ alpha RFLP subtypes appear to correlate with single, or associated HLA-DR specificities. Specific combinations of DQ beta with DQ alpha RFLPs also correlate with HLA-Dw splits of DR2 and DRw6. A system for HLA-DNA typing is described, which uses RFLP patterns generated by sequential hybridization of TaqI-digested DNAs with short DR beta, DQ beta and DQ alpha cDNA probes. The DQ beta and DQ alpha probes not only identify the DQ allele, but because of linkage disequilibrium with DR, help to assign the DR allele, which may not always be identified with a DR beta probe alone.

Acquisition of Paleolithic toolmaking abilities involves structural remodeling to inferior frontoparietal regions.

Human ancestors first modified stones into tools 2.6 million years ago, initiating a cascading increase in technological complexity that continues today. A parallel trend of brain expansion during the Paleolithic has motivated over 100 years of theorizing linking stone toolmaking and human brain evolution, but empirical support remains limited. Our study provides the first direct experimental evidence identifying likely neuroanatomical targets of natural selection acting on toolmaking ability. Subjects received MRI and DTI scans before, during, and after a 2-year Paleolithic toolmaking training program. White matter fractional anisotropy (FA) showed changes in branches of the superior longitudinal fasciculus leading into left supramarginal gyrus, bilateral ventral precentral gyri, and right inferior frontal gyrus pars triangularis. FA increased from Scan 1-2, a period of intense training, and decreased from Scan 2-3, a period of reduced training. Voxel-based morphometry found a similar trend toward gray matter expansion in the left supramarginal gyrus from Scan 1-2 and a reversal of this effect from Scan 2-3. FA changes correlated with training hours and with motor performance, and probabilistic tractography confirmed that white matter changes projected to gray matter changes and to regions that activate during Paleolithic toolmaking. These results show that acquisition of Paleolithic toolmaking skills elicits structural remodeling of recently evolved brain regions supporting human tool use, providing a mechanistic link between stone toolmaking and human brain evolution. These regions participate not only in toolmaking, but also in other complex functions including action planning and language, in keeping with the hypothesized co-evolution of these functions.

Curve -shift analysis of cryopreserved killer T cell function.

The conditions for cryopreservation and reconstitution after thawing of cytotoxic effector cells for cell mediated lympholysis (CML) tests are studied. Percentage recovery of functional activity is analysed not by the commonly used method of comparisons at a single concentration of effector cells but by the movement of the curve of functional activity on the axis of cell concentration in culture. By this method the position of the ascending slope of the dose-response curve, at several different effector to target cell ratios, is compared between fresh and frozen-thawed cells. Cooling rates giving optimal recovery were found to vary between experiments. Using simple techniques, optimal cooling rates were found to range from 0.3 to 1.0 degrees C/min, when using dimethyl sulphoxide (10% v/v). Dead cell debris, but not intact dead cells (which take up eosin), were shown to inhibit the lytic ability of functionally active frozen-thawed effector cells. This could be removed by centrifuging the thawed cells over Ficoll-Hypaque. In the recovered population the proportion of cells which excluded the vital dye, eosin, was greater than the proportion of functioning effector cells. This suggested that the cells which excluded eosin contained both functional and nonfunctional, partially damaged effector cells. Thus dye exclusion methods generally over-estimated the functional activity of thawed effector cells. When cells to be used as targets were preserved prior to treatment with phytohaemagglutinin (PHA) no abnormalities were detected in their behaviour to fresh cells.

Modified micromethod for detection of human Fc receptor-like material.

Soluble FcR-like material can be demonstrated in the culture supernatants of activated lymphocytes. We modified as assay for its detection which was based upon inhibition of complement dependent hemolysis, an unreliable phenomenon in our hands. The micromethod presented was developed using the culture supernatants of FcR producing and FcR non-producing lymphoblastoid cell lines. Suppression of hemolysis was consistently observed providing the assay was performed below 22 degrees C. It is suggested that the classical pathway of complement activation is inhibited by this material but not the alternative pathway. By this method FcR material can be detected in small cultures where cells are limited in number.

The predictive value of epitope analysis in highly sensitized patients awaiting renal transplantation.

The accumulation of highly sensitized patients (HSP) on renal transplant waiting lists is a problem faced by all transplant registries. We have studied the HLA class I serological reactivity of 20 random HSP and have related antibody specificity to primary amino acid sequence. In six patients we identified significant correlations (chi 2 test, r > or = 0.93) between panel reactivity and specific amino acid substitutions characteristic of HLA-A, -B, and -C public epitopes. Antibody reactivity was associated with up to three public epitopes in each patient. The 12 separate antibody specificities identified were associated with 10 residues. Seven correlated with HLA-A locus substitutions (Glu-62/Gly-65, Lys-66, Arg-114, His-114/Tyr-116/Lys-127, Thr-142/His-145 [x2], and Thr-149), two with HLA-B locus substitutions (Thr-24, Ser-24) and three with interlocus antibodies associated with either HLA-A and B (Leu-82/Arg-83 [x2]) or with HLA-B and -C substitutions (Leu-163). This information allowed us to predict HLA class I allelic products of known primary sequence that would react negatively with each HSP serum. Windows of acceptable mismatches (WAMMs) can thus be delineated with a view to crossmatch negative transplantation without the need for exhaustive serological analysis. Surprisingly we found that WAMMs for these patients included up to 80% of the 10 commonest HLA class I haplotypes in the British population with four patients being crossmatch compatible with A1,3; B7,8. These observations lead us to propose a more intelligent approach to transplanting HSP based on epitope analysis and definition of WAMMs.

The variation among transplant center results in the United Kingdom and Ireland from 1977 to 1981.

An analysis of pooled data from transplants performed between 1977 and 1981 in 29 centers throughout the United Kingdom and Ireland revealed that the pattern of loss varied according to cause and postoperative time. Loss from rejection was characterized by a bimodal pattern in which early (0-25 days) and late (26-100 days) peaks of rejection were distinguishable. Rejected second transplants exhibited this phenomenon more than first transplants, and the loss was proportionately greater during the early period, suggesting that prior sensitization played an important role. Graft loss from technical causes and recipient death showed distinctly different patterns of loss. These findings suggested that, when possible, transplant survival statistics should be analyzed separately according to postoperative time and cause of loss. In applying these preliminary observations of the pooled data to a comparative study of the results in the different centers it was noted that such comparisons could be substantially affected by random variability in estimates of actuarial survival rates. Therefore, a simple method of ranking was developed in which centers were allocated to high or low survivorship categories, or to an indeterminate category when the standard error in estimated actuarial survival was relatively large. Whereas the variation in loss rate from death with a functioning transplant (DWFT) was found to be indistinguishable from random variability, both nonimmunological failure (NIF) and immunological failure (IF) of the graft were found to be legitimate bases for ranking. Furthermore, center ranking based on IF at 0-25 days failed to exhibit a significant relationship with IF at 26-100 days, which could indicate important center differences associated with antirejection treatments during these two periods. These results showed that, ideally, time-cause parameters should be analyzed separately when comparing transplant survival statistics in different centers.

Predicting match grade and waiting time to kidney transplantation.

We show how to estimate expected waiting time to transplantation and match grade potential for each patient awaiting kidney transplantation on a multicenter waiting list. We predict that some easily well-matched patients may be unlikely to receive a well-matched graft through accepting an earlier offer of a poorly matched kidney. Other patients, who are difficult to match, may be unlikely to receive even a poorly matched kidney within a reasonable time.

Analyzing transplant survival data.

Stratified proportional hazards regression is described as a method of estimating multifactorially preoperative factor effects on graft survival--and, at the same time, making due allowances for unknown transplant-center-specific influences. The multifactorial aspect of the method overcomes the biases inherent in analyzing transplant survival data one factor at a time, and stratification allows the data from many centers to be used simultaneously without the dangers associated with simple pooling of data from many sources. The proportionality and regression assumptions implicit in the method enable the data to be used efficiently, but must be validated on the data. Methods by which these assumptions may be relaxed are described--in particular, the stratified piece-wise proportional hazards regression method.

Absence of tolerance to noninherited maternal transplantation antigens expressed on skin and cornea.

Tolerance to noninherited maternal antigens (NIMAs) of the MHC was investigated in a rat model involving both skin and corneal transplants. Recipient animals were obtained by backcrossing F1 hybrids to parental strain animals. In one group of experiments, crosses were (DA[RT1a] x LEW[RT1(1)]) female-to-LEW male and, in a second group, (DAxPVG[RT1c]) female-to-PVG male. Homozygous backcross offspring (RT1(1/1) or RT1c/c) were putatively tolerant to DA NIMAs if the mother was a hybrid animal, having been exposed to these antigens in utero. The equivalent offspring of hybrid fathers, i.e., LEW female x (DAxLEW) male or PVG female x (DAxPVG) male, served as putatively nontolerant controls. Hemagglutinating antibody levels were measured against the class I RT1Aa antigen on days 7 and 14 after up to three consecutive subcutaneous DA strain skin grafts. Significantly lower titers were found in the putatively tolerant group 7 days after the first skin graft in the RT1a-to-RT1(1) combination (P < 0.05). Levels were not significantly different at any other time point, or at any time point in the RT1a-to-RT1c combination. Tolerance to a corneal graft was not demonstrated in either the strongly rejecting RT1a-to-RT1(1) combination or weakly rejecting RT1a-to-RT1c, whether or not animals were presensitized to RT1a antigens with DA skin grafts. We conclude that tolerance to NIMAs is unimportant in this clinical rat model of transplantation.

Renal transplant rejection. Transient immunodominance of HLA mismatches.

Many studies have shown long-term benefits of HLA matching in kidney transplantation. By examining rates of graft loss within consecutive posttransplant intervals, using data from the UK Transplant Service, we show that the long-term benefits of HLA matching are due to reduction of the graft failure rate within five months of transplantation. After 5-months, HLA-A,B,DR matching appears to have little impact on graft loss. We suggest that graft losses after 5 months may be attributable to non-HLA targets.

Substantial benefits of tissue matching in renal transplantation.

The purpose of this study was to perform a rigorous statistical analysis of the benefits of HLA-A,B and DR matching in renal transplantation. Graft survival in 2282 first cadaver kidney transplants, recorded and followed up by the United Kingdom Transplant Service (UKTS), was analyzed using the piecewise proportional hazards regression method. The results show that substantial improvements in graft survival are obtained when there is DR compatibility and at most one A or B mismatch, but that there is little advantage in tissue matching unless this degree of matching can be attained. So far, few graft recipients have benefited substantially through tissue matching (24% of kidneys exchanged through UKTS in 1984). This is partly attributable to unresolved technical problems in DR typing. However simulations show that under ideal conditions, with a pool of 3000 patients awaiting transplantation, considerable improvements in graft survival can be obtained in over 60% of recipients.

Immunogenetic and clinical factors affecting renal transplantation. A rigorous analysis of data recorded by the UK Transplant Service.

A total of 3653 first cadaver kidney transplants in nondiabetic recipients over 15 years of age, performed in 30 transplant centers throughout the United Kingdom between 1978 and 1983, were analyzed to discover which of many recorded recipient, donor, surgical, and tissue-matching variables were important for graft survival, and in which postoperative period they exerted their maximum influence. A considerable effort was invested in checking the validity of the data and the appropriateness of the statistical methods. The duration of dialysis prior to transplantation was associated with a substantially reduced risk of graft failure, particularly at later postoperative times. Good HLA-B locus matching was also found to enhance graft survival--but, by contrast, HLA-A locus matching showed no significant effect on survival. Recipient age over 45 years, high serum reactivity, and grafts with anoxia time (the interval between circulatory arrest and perfusion with ice) exceeding 10 min were found to be associated with poor graft survival. Trends in survival were identified across calendar years of transplant, such that early graft failure (0-15 days) had increased in recent years, whereas later graft failure (greater than 15 days) had declined.

UHG crossmatching. A comparison with PCR-SSO typing in the selection of HLA-DPB1-compatible bone marrow donors.

The technique of universal heteroduplex generator (UHG) crossmatching has been developed to permit comparison of HLA-DPB1 alleles between two or more individuals. It offers a rapid and simple method of screening prospective bone marrow donors for HLA-DPB1 compatibility with the recipient. We present the nucleotide sequence and describe the method of construction of the DPB1 UHG. To test its effectiveness, 56 patient-bone marrow donor pairs previously HLA-DPB1-typed by PCR-SSO probing, were tested by UHG crossmatching. In 52/56 (93%) pairs there was concordance between PCR-SSO typing and UHG crossmatching. All 32 pairs that were defined as mismatched by PCR-SSO typing were also mismatched by UHG crossmatching. We conclude that UHG crossmatching is a simple, sensitive, and cost effective method of HLA-DPB1 matching for the rapid selection of compatible bone marrow donors.

Influence of donor and histocompatibility factors on corneal graft outcome.

The Corneal Transplant Follow-up Study has followed 2311 penetrating keratoplasties for up to 450 days after transplant. A total of 207 failures were observed, including 65 classical rejections and 35 endothelial decompensations. At 12 months, graft survival was 89%, and survival free from rejection was 87%. For surviving grafts, risk of failure reduced from 4.8% in the first 75 days and stabilized after 5 months at 1.2% in each 75-day interval. Risk of rejection initially followed a similar pattern, but then increased after 12 months. Multifactorial analyses accounted for differences in recipient characteristics and interrelationships of donor factors. Donor age, sex, cause of death, and method of corneal storage were not found to influence significantly either time to graft failure or time to first rejection. Grafts in prospectively tissue-typed donor-recipient pairs were generally considered before surgery to be at increased risk of either graft failure or rejection. With due allowance, increasing risk of rejection was associated with increasing numbers of mismatches at HLA-A and HLA-B broad antigens. The opposite was true at HLA-DR broad antigens, where increased risk of rejection was observed with no mismatches.

A DNA-RFLP typing system that positively identifies serologically well-defined and ill-defined HLA-DR and DQ alleles, including DRw10.

A single enzyme/multiple probe system of HLA-DR and DQ typing using restriction fragment-length polymorphism (RFLP) analysis is presented. TaqI-digested genomic DNAs are hybridized sequentially with short DR beta, DQ beta, and DQ alpha cDNA probes. The DR beta probe discriminates between the DR allelic specificities DR1 to DRw14, with the two exceptions of some DR3/DRw13 and some DR7/DRw9 combinations. We describe the positive identification of a DRw10-specific RFLP and demonstrate its segregation in families. The DQ beta probe defines an allelic system that identifies the alleles DQw1, DQw2, and DQw3. This permits the resolution of DR3/DRw13 and DR7/DRw9 alleles by defining the DR/DQ association caused by linkage disequilibrium. The DQ alpha probe defines another allelic series interrelated with, but independent from, the DQ beta series. Specific DQ beta/DQ alpha RFLP combinations correlate with known Dw splits of DR2, DRw6, and DR7. Combined use of the three probes permits the identification of HLA-DR, DQ, and certain Dw specificities and provides an effective and easily interpretable system for major histocompatibility complex class II allogenotyping.

The role of HLA matching in transplantation.

All viable human tissues transplanted from one individual to another are at risk of rejection. The extent of risk depends on the donor HLA-host T cell receptor disparity. Such disparity is now known to involve genetic products coded by both HLA and non-HLA genes. T cell responses to mismatches on transplants are graded: the greater the mismatch the greater the number of clones of T cells responding. Consequently, the more severe the rejection process. Global statistics support this view in that cumulative mismatches at HLA are associated with poorer graft survival. However such studies also suggest that mismatches at different HLA loci vary in potency. The strength of mismatch seems to increase from HLA-A, the weakest, through HLA-B to HLA-DR, the most potent. Analysis of clinical results suggests that the risk associated with HLA mismatches in kidney transplantation dwindles after the first five months post-transplant. Although graft losses tend to occur sporadically thereafter, no major risk associated with HLA mismatches can be discerned. Whether this dwindling impact of HLA mismatches with time post-transplant is a general phenomenon applicable to all transplants, or whether it suggests some form of adaptation of host to graft in kidney transplant recipients alone is a subject for further exploration. Tissues vary widely in their susceptibility to rejection through HLA mismatches.(ABSTRACT TRUNCATED AT 250 WORDS)