Fuzzy pattern classification of hemodynamic data can be used to determine noninvasive intracranial pressure.

OBJECTIVE: The authors previously introduced a method in which intracranial pressure (ICP) was estimated using parameters (TCD characteristics) derived from cerebral blood flow velocity (FV) and arterial blood pressure (ABP). Some results suggested that this model might be influenced by the patient's state of cerebral autoregulation and other clinical parameters. Hence, it was the aim of the present study to improve the method by modifying the previously used global procedure in certain subgroups of patients. METHODS: In 103 traumatic brain injured patients (3-76 years, mean: 31 +/- 16 years) signal data of FV, ABP and ICP were used to generate samples of TCD characteristics together with time corresponding ICP. Fuzzy Pattern Classification was used to identify cluster subsets (classes) of the sample space. On each class a local estimator of ICP was defined. This approach provides a non-invasive assessment of ICP (nICP) as follows: Using FV and ABP the TCD characteristics were computed and related to the matching classes. nICP was calculated as a weighted sum of local ICP estimations. RESULTS: ICP A and B waves and long-term trends could be visibly assessed. The median absolute difference between ICP and nICP was 5.7 mmHg. CONCLUSIONS: The class structure of the model facilitates nICP assessment in heterogeneous patient groups and supports a stepwise extension of the target patient group without affecting the former validity.

Positive outcomes in traumatic brain injury-vegetative state: patients treated with bromocriptine.

OBJECTIVE: To assess the effects of multidisciplinary rehabilitation interventions and use of bromocriptine on outcome in patients with traumatic brain injury-vegetative state (TBI-VS). DESIGN: Retrospective review of clinical cases. SETTING: Free-standing rehabilitation hospital; Acute and extended rehabilitation hospital. PARTICIPANTS: Five consecutive TBI-VS patients, as well as 33 TBI-VS patients and 37 traumatic brain injury-minimally conscious state (TBI-MCS) patients reported in the literature. INTERVENTIONS: Bromocriptine administration, systematic neuropsychologic testing, sensory stimulation, and traditional comprehensive rehabilitation with physical therapy, occupational therapy, and speech therapy. MAIN OUTCOME MEASURES: Disability Rating Scale (DRS) at 1, 3, 6, and 12 months postinjury and FIM instrument scores at 1 month and 12 months postinjury, Coma Recovery Scale, and Barry Rehabilitation Inpatient Screening of Cognition. RESULTS: The 5 TBI-VS patients emerged from a VS into a MCS and regained functional status. Their recovery of physical and cognitive functioning, as rated by the DRS, was greater than previously reported in the literature for patients in a VS or MCS at 3, 6, and 12 months postinjury. CONCLUSION: Bromocriptine administration, systematic neuropsychologic testing, sensory stimulation, a comprehensive rehabilitation program, or a combination of these treatments may enhance functional recovery in this TBI-VS patient group. Further systematic study to quantify the contribution of these variables and to reproduce this data in a larger patient population should be performed.

Association of soluble HLA-G plasma levels with HLA-G alleles.

Soluble HLA-G (sHLA-G) molecules are found in the peripheral blood of healthy females and males, in cord blood and in amniotic fluids and discussed to be a mediator in maternal-fetal tolerance. In this study we investigated whether there are allele-specific differences in expression of sHLA-G molecules. For this, the sHLA-G plasma concentrations of 94 healthy unrelated individuals were measured by ELISA and correlated to their HLA-G genotypes, as determined by sequence analysis of exon 2 and 3 of the HLA-G gene. Mean sHLA-G levels in individuals with the most common HLA-G alleles G*01011 (27.0+/-2.1 SEM ng/ml, n=66), G*01012 (28.4+/-3.2 SEM ng/ml, n=34) were very similar. In contrast, individuals carrying the HLA-G*01013 (8.1+/-1.7 SEM ng/ml, n=17) or the "null" allele HLA-G*0105N (8.2+/-3.2 SEM ng/ml, n=7) presented significantly (P(c)=0.001 and P(c)<0.01, resp.) reduced sHLA-G levels. Furthermore, individuals with the HLA-G*01041 allele had significantly (P(c)=0.004) increased sHLA-G levels (42.5+/-4.6 SEM ng/ml, n=14). These results demonstrate that the generation of sHLA-G molecules is associated to certain HLA-G alleles and imply that sHLA-G levels are under genetic control. As low and high sHLA-G plasma levels did not segregate with HLA haplotypes including the HLA-G*01013 or *01041 allele, additional mechanisms may be involved in the regulation of the individual sHLA-G levels. Nevertheless, the existence of "low" and "high secretor" HLA-G alleles further suggests different levels of functionality in immune regulation.

Immune response to perforated and partially demineralized bone allografts.

Immune responses have been shown to be involved in the pathogenesis of clinical complications of cortical bone allografts. In an attempt to reduce the immunogenicity of these allografts, we evaluated cortical bone allografts modified by laser perforation and partial demineralization transplanted orthotopically into sheep tibiae. The recipient animals were divided into three groups, of eight animals each, according to the type of cortical allograft that was transplanted: group 1, no treatment (control); group 2, demineralization only; and group 3, laser perforation and partial demineralization. All animals were tissue-typed by biochemical definition of MHC class I molecules, using unidimensional isoelectric focusing and Western blotting. Mismatches of donors and recipients were assessed by testing samples of each donor and recipient pair in parallel and by comparing their individual bands. Donor-specific alloantibodies were detected by a similar technique, using an enzyme-linked immunosorbent assay (ELISA) format. Negative controls were included in all tests. All grafts were poorly immunogenic, whether they were untreated, processed by partial demineralization, or processed by both laser perforation and partial demineralization. Only two recipient animals showed a transient, antibody-mediated donor-specific immune response. One of these animals had received a control allograft, whereas the other animal had received a laser-perforated and partially demineralized bone allograft. All of the grafts in this study, including control grafts, were stripped of soft tissues and their bone marrow was removed; cellular sources of alloantibody stimulation may have been eliminated by these processes. The results of this study suggest that immune responses to bone allografts may be reduced by removing the bone marrow and adjacent soft tissues. The processing of cortical bone allografts by laser perforation and partial demineralization appeared to have little effect on immune responses.

Soluble HLA levels in early pregnancy after in vitro fertilization.

Intact pregnancy can be interpreted as a state of maternal immunotolerance toward an haploidentical fetus. Soluble HLA (sHLA) molecules increase during episodes of allograft rejection and are discussed as candidates to modulate immune responses. We questioned whether after in vitro fertilization (IVF) the subsequent intact pregnancy, early abortion, or tubal pregnancy influence the courses sHLA serum levels. Therefore, serum samples of 65 IVF patients were assayed by ELISA for sHLA-I, sHLA-G, and sHLA-DR concentrations preovulatorily and after a positive HCG test weekly until the 9th gestational week (GW). In 20 patients experiencing an early abortion the preovulatory sHLA-G mean level of 25.9 +/- 3.9 SEM ng/ml and the share of 4.2 +/- 0.8 SEM % on total sHLA-I were significantly (p < 0.05) reduced compared to women with intact pregnancy. The same differences (p < 0.0001) were seen during the monitoring of sHLA-G and sHLA-I levels in intact pregnancy versus early abortion until 9th GW. Twin pregnancy revealed a drastically increase of sHLA-G levels from the 8th GW compared to singleton pregnancies. Further, individual sHLA-DR levels increased during intact pregnancy but decreased in the group of early abortion. With regard to sensitivity and specificity for pregnancy outcome sHLA quantitation reached similar weight as routine HCG determinations at GW 5. Especially women with preovulatory low sHLA-G levels appear to be on risk for early abortion after IVF.

Monitoring of soluble HLA class I size variants after liver transplantation.

To monitor soluble HLA class I (sHLA-I) and their size variants after liver transplantation (LTX) plasma samples from 22 LTX patients were studied by sHLA-I ELISA, SDS-PAGE, and densitometry. Samples collected were classified into three groups: Group 1 comprised samples taken during episodes without complications, group 2 during episodes of cholangitis/cholestasis (CC), and group 3 during episodes of acute rejection (AR). Compared to group 1 (0.27 +/- 0.03 SEM microg/ml) mean sHLA-I increments in groups 2 and 3 were with 0.53 +/- 0.05 SEM microg/ml and 0.47 +/- 0.04 SEM microg/ml increased (p < 0.001). The same samples were studied by SDS-PAGE and the 43, 39, and 35 kD sHLA-I variants were quantified densitometrically. In samples of group 1 ratios of 43 vs. 39 kD bands revealed a mean of 2.1 +/- 0.3, whereas in group 2 and 3 these were only 0.8 +/- 0.1 SEM and 0.9 +/- 0.1 SEM, respectively, (p < 0.001). For the relation between 43 and 35 kD variants a reduced ratio of 1.1 +/- 0.2 SEM was confined to group 3 samples (p < 0.001), as groups 1 and 2 had ratios of 13.4 +/- 2.3 SEM and 8.4 +/- 2.9 SEM, respectively. This indicates that elevated sHLA-I levels during CC or AR are mainly caused by increases of 39 and/or 35 kD sized molecules. Therefore, our study demonstrates, that after LTX the contribution of sHLA-I size variants to total sHLA-I amounts changes drastically during immune activation pointing to different mechanisms of sHLA-I release.

Detection of soluble HLA-G molecules in plasma and amniotic fluid.

Although the cDNA sequence of HLA-G antigens is compatible with their expression as soluble molecules (sHLA-G), the determination of native sHLA-G levels in body fluids has not yet been described. The lack of this information is likely to reflect the difficulties in developing an assay suitable to measure sHLA-G antigens in the presence of soluble HLA-A, -B and -C (sHLA-I) antigens, since most of the available anti-HLA-G mAb do not detect soluble beta2-m associated HLA-G antigens or crossreact with sHLA-I antigens. Therefore, we have developed a two-step assay which eliminates the interference of classical HLA class I antigens. In the first step, the sample is depleted of sHLA-I antigens and of HLA-E antigens with mAb TP25.99. Then, HLA-G antigens are captured with mAb W6/32 and detected with anti-beta2-m mAb in ELISA. Utilizing this assay, sHLA-G antigen levels were measured in EDTA plasma from 92 controls with known HLA types, 28 women at delivery and the corresponding cord bloods and in 50 amniotic fluids. Mean sHLA-G plasma levels did not differ between males (24.9+/-3.0 SEM ng/ml; n=42) and females (20.1+/-2.1 SEM ng/ml; n = 50). However, sHLA-G levels in HLA-A11 positive probands (mean: 13.0+/-4.4 SEM ng/ml; n=12) were significantly (P<0.05) lower than in HLA-A11 negative ones (mean: 24.5+/-2.0 SEM ng/ml; n=80). sHLA-G levels in women at delivery (mean: 22.9+/-2.2 SEM ng/ml; n=28) were in the range of controls but were significantly (P<0.001) reduced in the corresponding cord bloods (mean: 13.8+/-1.5 SEM ng/ml; n=28). sHLA-G levels in amniotic fluids (mean: 15.5 + 1.0 SEM ng/ml; n=50) were significantly (P<0.001) lower than in plasma. sHLA-G levels were 5 and 11% of those of sHLA-I antigens in plasmas and amniotic fluids, respectively. Individual sHLA-G levels were not correlated with sHLA-I levels. SDS-PAGE analysis of plasma sHLA-G antigens revealed two molecular variants with a 35 kD and a 27 kD MW corresponding to the sizes of sHLA-G1 and -G2 isoforms. In conclusion, our study has shown that the two-step assay we have developed is reliable in measuring sHLA-G antigen levels. This assay will facilitate the analysis of the biological and clinical significance of sHLA-G antigens in plasma.

Detection efficacy of soluble HLA-A, B antigens using 1D-IEF.

Soluble HLA class I alloantigens (sHLA class I) can be typed according to their isoelectric points (IEP) after immunoprecipitation by w6/32 monoclonal antibody (mAb) coupled to immunomagnetic beads and focusing. In order to prove the large scale efficacy of this methodology, EDTA-plasma samples from 344 probands HLA-A, B typed by serology were analysed by one-dimensional isoelectric focusing and HLA class I specific Westernblot (1D-IEF). In addition, detergent solubilized HLA class I membrane molecules from approximately one half of the probands were studied too. Soluble HLA-A24,B7,B18,B62 antigens were identified in nearly all experiments, whereas A28, B13, and B51 could be detected in about 50%. A third group of HLA antigens (A26, B8, B44) could be visualized rarely. The difficulties of detection might be due to the different affinity of mAb w6/32 to certain sHLA class I gene products or to variable amounts of sHLA class I in the plasma specimens. Some modifications of the antigen capture technique have already led to a slightly better degree of antigen recognition in 25 probands tested. Thus, HLA-A, B typing using sHLA molecules and 1D-IEF in the assay format presented does not yet seem to be a definitive alternative for HLA class I serology or biochemistry of membrane-bound HLA class I molecules but it should be a promising technique if no cells are available or donor-derived sHLA allotypes are to be monitored after HLA mismatched organ transplantation.

Segregation study of the soluble 39-kD HLA class I heavy chain.

To study the variable expression and inheritance of the 39-kD M(r) form of HLA class I molecules, we investigated the plasma of 12 families with 61 members and 41 unrelated individuals by SDS gel electrophoresis and class-I-specific immunoblotting. Seven families were informative for the presence and absence of the 39-kD band with a clear segregation pattern in coupling with HLA. The data were in agreement with a codominant expression of this molecular variant of sHLA class I. Of the 48 independent HLA haplotypes, 19 were identified as carrying the 39-kD deficiency. However, there was no clear association with HLA-B7 or gender. Under the assumption that all haplotypes in the five non-informative families carried the expressed 39-kD variant, approximately 40% of the Caucasian population should be heterozygous deficient for the 39-kD band of sHLA class I.