Pretransplant IgG reactivity to apoptotic cells correlates with late kidney allograft loss.

Preexisting serum antibodies have long been associated with graft loss in transplant recipients. While most studies have focused on HLA-specific antibodies, the contribution of non-HLA-reactive antibodies has been largely overlooked. We have recently characterized mAbs secreted by B cell clones derived from kidney allograft recipients with rejection that bind to apoptotic cells. Here, we assessed the presence of such antibodies in pretransplant serum from 300 kidney transplant recipients and examined their contribution to the graft outcomes. Kaplan-Meier survival analysis revealed that patients with high pretransplant IgG reactivity to apoptotic cells had a significantly increased rate of late graft loss. The effect was only apparent after approximately 1 year posttransplant. Moreover, the association between pretransplant IgG reactivity to apoptotic cells and graft loss was still significant after excluding patients with high reactivity to HLA. This reactivity was almost exclusively mediated by IgG1 and IgG3 with complement fixing and activating properties. Overall, our findings support the view that IgG reactive to apoptotic cells contribute to presensitization. Taking these antibodies into consideration alongside anti-HLA antibodies during candidate evaluation would likely improve the transplant risk assessment.

Polyreactive antibodies developing amidst humoral rejection of human kidney grafts bind apoptotic cells and activate complement.

Antibody mediated rejection (AMR) is associated with a variety of graft-reactive antibodies following kidney transplant. To characterize these antibodies, we immortalized 107 B cell clones from a patient with AMR. In a previous study, we showed that six clones were reacting to multiple self-antigens as well as to HLA and MICA for two of them, thus displaying a pattern of polyreactivity. We show here that all six polyreactive clones also reacted to apoptotic but not viable cells. More generally we observed a nearly perfect overlap between polyreactivity and reactivity to apoptotic cells. Functionally, polyreactive antibodies can activate complement, resulting in the deposition of C3d and C4d at the surface of target cells. Testing the serum of 88 kidney transplant recipients revealed a significantly higher IgG reactivity to apoptotic cells in AMR patients than in patients with stable graft function. Moreover, total IgG purified from AMR patients had increased complement activating properties compared to IgG from non-AMR patients. Overall, our studies show the development of polyreactive antibodies cross-reactive to apoptotic cells during AMR. Further studies are now warranted to determine their contribution to the detection of C4d in graft biopsies as well as their role in the pathophysiology of AMR.

Expansion of polyreactive B cells cross-reactive to HLA and self in the blood of a patient with kidney graft rejection.

Antibody rejection is often accompanied by nondonor HLA specific antibodies (NDSA) and self-reactive antibodies that develop alongside donor-specific antibodies (DSA). To determine the source of these antibodies, we immortalized 107 B-cell clones from a kidney transplant recipient with humoral rejection. Two of these clones reacted to HLA class I or MICA. Both clones were also reactive to self-antigens and a lysate of a kidney cell line, hence revealing a pattern of polyreactivity. Monoclonality was verified by the identification of a single rearranged immunoglobulin heavy chain variable region (VH) sequence for each clone. By tracking their unique CDR3 sequence, we found that one such polyreactive clone was highly expanded in the patient blood, representing ~0.2% of circulating B cells. The VH sequence of this clone showed evidence of somatic mutations that were consistent with its memory phenotype and its expansion. Lastly, the reactivity of the expanded polyreactive B-cell clone was found in the patient serum at time of rejection. In conclusion, we provide here proof of principle at the clonal level that human antibodies can cross-react to HLA and self. Our findings strongly suggest that polyreactive antibodies contribute to DSA, NDSA as well as autoantibodies, in transplant recipients.

Chronic humoral rejection of human kidney allografts is associated with MMP-2 accumulation in podocytes and its release in the urine.

Chronic humoral rejection (CHR) is an important cause of late graft failures following kidney transplantation. Overall, the pathophysiology of CHR is poorly understood. Matrix metalloproteinase-2 (MMP-2), a type IV collagenase, has been implicated in chronic kidney disease and allograft rejection in previous studies. We examined the presence of MMP-2 in allograft biopsies and in the urine of kidney transplant recipients with CHR. MMP-2 staining was detected by immunohistochemistry in podocytes for all CHR patients but less frequently in patients with other renal complications. Urinary MMP-2 levels were also significantly higher in CHR patients (median 4942 pg/mL, N = 27) compared to non-CHR patients (median 598 pg/mL, N = 65; p < 0.001). Elevated urinary MMP-2 correlated with higher levels of proteinuria in both CHR and non-CHR patients. Longitudinal analysis indicated that increase in urine MMP-2 coincided with initial diagnosis of CHR as documented by the biopsies. Using an enzymatic assay, we demonstrated that MMP-2 was present in its active form in the urine of patients with CHR. Overall, our findings associate MMP-2 with glomerular injury as well as interstitial fibrosis and tubular atrophy observed in patients with CHR.

Discrete Structure of the Brain Rhythms.

Neuronal activity in the brain generates synchronous oscillations of the Local Field Potential (LFP). The traditional analyses of the LFPs are based on decomposing the signal into simpler components, such as sinusoidal harmonics. However, a common drawback of such methods is that the decomposition primitives are usually presumed from the onset, which may bias our understanding of the signal's structure. Here, we introduce an alternative approach that allows an impartial, high resolution, hands-off decomposition of the brain waves into a small number of discrete, frequency-modulated oscillatory processes, which we call oscillons. In particular, we demonstrate that mouse hippocampal LFP contain a single oscillon that occupies the θ-frequency band and a couple of γ-oscillons that correspond, respectively, to slow and fast γ-waves. Since the oscillons were identified empirically, they may represent the actual, physical structure of synchronous oscillations in neuronal ensembles, whereas Fourier-defined "brain waves" are nothing but poorly resolved oscillons.

Phase I/pharmacokinetic reevaluation of thioTEPA.

Because the initial evaluation of N,N',N''-triethylenethiophosphoramide (thioTEPA) preceded the standardized approach to the Phase I trials, uncertainty surrounds the recommended dose. Since it has recently been demonstrated that an almost 100-fold increase in dose can be administered in bone marrow transplant regimens, we conducted a Phase I reevaluation of thioTEPA. ThioTEPA was administered i.v. in 50 ml 5% dextrose in water over 10 min. Twenty-seven patients were entered at doses ranging from 30 to 75 mg/m2. The major toxic effect was myelosuppression; thrombocytopenia greater than or equal to grade 3 occurred in four of seven patients, and leukopenia greater than or equal to grade 3 in two of seven patients at 75 mg/m2. Among eight patients at 65 mg/m2 only two had greater than or equal to grade 3 myelosuppression making this the recommended new phase II dose for the majority of patients. Moderate (grade 2) easily controlled nausea and vomiting was the only other major side effect. There was no alopecia or mucosal or neurological toxicity. Three partial remissions were observed among nine previously treated ovarian cancer patients. Plasma concentrations of thioTEPA and its major active metabolite triethylenephosphoramide (TEPA) were measured by gas chromatography. The half-life of thioTEPA ranged from 51.6 to 211.8 min, and its pharmacokinetics was dose dependent; total body thioTEPA clearance decreased with increasing dose. The half-life of TEPA was considerably longer than that of the parent compound (3.0 to 21.1 h); as a result, the area under the plasma concentration-time curve (AUC) of TEPA was severalfold greater than that of the parent compound. The ratio of TEPA AUC to thioTEPA AUC decreased with increasing dose, suggesting that formation of TEPA is a saturable step in elimination. The AUC and total body clearance of thioTEPA, but not of TEPA, were closely correlated with neutrophil but not platelet toxicity.

Specificity of antitermination mechanisms. Suppression of the terminator cluster T1-T2 of Escherichia coli ribosomal RNA operon, rrnB, by phage lambda antiterminators.

Transcription of the ribosomal RNA operons (rrn) in Escherichia coli is subject to an antitermination mechanism whereby RNA polymerase is modified to a termination-resistant form during transit through the rrn leader region. This antitermination mechanism is unable to overcome the T1-T2 terminator cluster located at the end of an rrn operon, such as rrnB. We have tested the specificity with which the T1-T2 terminators override an antitermination mechanism, by placing the terminator cluster downstream from the nut and qut sites recognized by phage lambda N and Q gene antiterminators, respectively. Measurement of downstream gene expression shows that RNA polymerase modified by either N or Q reads through the T1-T2 terminators quite efficiently. This supports the view that T1-T2 are not superterminators, and that the rrn antitermination mechanism may have a restricted terminator specificity.

Effect of formulary restriction of cefotaxime usage.

Cefotaxime sodium was assigned to the open formulary for 12 months and then was placed on formulary restriction to evaluate the restriction's effect on rate of use by services and appropriateness of use. Over 18 months, 187 cases (72 before and 115 after restriction) were reviewed. The majority of use (prerestriction and postrestriction) was in the medicine, pediatrics, and surgery services. The postrestriction usage rate for the three services increased significantly. Cefotaxime was used appropriately in 85% of cases during both periods and was not used prophylactically. Appropriateness of use was independent of formulary restriction. During both periods, approximately 76% of patients received cefotaxime for pneumonia, sepsis, meningitis, or immunosuppression. Of 205 infections, gram-negative bacilli accounted for over half of the pathogens isolated. Thus, formulary restriction was ineffective in reducing the rate of cefotaxime usage and had no effect on the appropriateness of usage.

Oral ciprofloxacin in the treatment of serious soft tissue and bone infections. Efficacy, safety, and pharmacokinetics.

Forty-eight patients were enrolled in a clinical study of oral ciprofloxacin for the treatment of soft tissue or bone infections. Patients received 500 to 750 mg of ciprofloxacin every 12 hours. In the predominantly older population studied, there were 13 patients with osteomyelitis, 24 diabetic patients with soft tissue infection and probable osteomyelitis, and 11 patients with other soft tissue infections. Infecting pathogens included Pseudomonas aeruginosa in 25 patients, Serratia species in nine patients, Staphylococcus aureus in 13 patients, and other aerobic gram-negative rods in 21 patients. Clinical response (defined as resolution or improvement) was noted in 84 percent of patients with non-diabetic osteomyelitis, in 79 percent of patients with diabetic infections, and in 91 percent of patients with soft tissue infections. Microbiologic outcome was very favorable in 75 percent of cases, and Pseudomonas responded as well as any other pathogen. Pharmacokinetic properties of ciprofloxacin were evaluated in 12 patients, and the data were analyzed using both compartmental and non-compartmental analyses. Mean values for compartmental rate constants (hours-1) were as follows: absorption rate constant = 1.15; intercompartmental rate constants, k12 = 0.48, and k21 = 0.58; elimination rate constant = 0.46; distribution rate constant = 1.31; and terminal elimination rate constant = 0.19. The apparent volume of distribution at steady state/bioavailability was 196 liters and total body clearance/bioavailability was 45.9 liters/hour. The mean time to peak concentration was 1.3 hours. The mean peak concentration as determined by compartmental fitting (2.4 micrograms/ml) underestimated the observed peak (3.2 micrograms/ml) by 24.8 percent. Clearance of ciprofloxacin was similar regardless of the method used to fit the data, whereas the volume of distribution was significantly different when the two analysis techniques were compared. Ciprofloxacin was well tolerated, with the most frequent adverse reactions being rash, gastrointestinal intolerance, and increased levels of liver enzymes, each of which occurred in five patients.

Afferent projections to the hypothalamic area controlling emotional responses (HACER).

The Mesulam technique for horseradish peroxidase was used to study the subcortical afferent projections to a location in the hypothalamus that has been shown to control the complete cardiovascular (CV) response accompanying a specific emotional behavior. Major projections common to all baboons injected included the lateral septal nucleus; medial, cortical and basal amygdala; the anteroventral third ventricle area; the preoptic areas; the subiculum; the paraventricular nucleus of the thalamus; periventricular gray and the central gray of the midbrain; the midbrain tegmentum; locus ceruleus, parabrachial and raphe cells in the pons; and in the medulla, raphe nuclei, the nucleus of the solitary tract, in and around the dorsal motor nucleus of the vagus, and in the region of the nucleus ambiguus. Other projections in some but not all baboons included the subfornical organ and the midline and dorsomedial nuclei of the thalamus. The nucleus of the diagonal band of Broca was labeled to some degree with all injections but was most heavily labeled with the injection extending more laterally in the hypothalamus. These results fit well with physiological and behavioral studies dealing with neural control of emotional and CV responses and support the concept of an integrative area in the hypothalamus concerned specifically with the control of CV response accompanying emotion.

Origin of the pyramidal tract determined with horseradish peroxidase.

The origin of the axons contained in the pyramidal tract (PT) of the cat was established using retrograde transport of horseradish peroxidase (HRP). A complete section was made through a PT at the level of the medulla oblongata and HRP was applied to the sectioned axons. Cat brains were cut in frontal and sagittal planes and HRP-labeled cells were plotted in outlines of the brain sections. The entire cortical region containing PT cells was divided into 8 subregions and the percent of PT cells was determined in each. Surface cortex, subregions 1, 3 and 8, contained only 30--40% of PT cells; the majority resided in deep sulcal cortex, in subregions 2, 4, 5, 6 and 7. Subregion 1 (containing 6--12% of PT cells) extends rostral to the cruciate sulcus; subregion 3 (15--22%) extends from the cruciate sulcus caudally to the ansate sulcus; subregion 8 (7--8%) covers cortex laterally adjacent to subregion 3. The hidden banks of the cruciate sulcus contained the greatest concentration of PT cells, 28--34% in the dorsal bank (subregion 5) and 15--20% in the ventral bank (subregion 4). The coronal sulcus contained only 2--5% of PT cells in its dorsal bank (subregion 6) and 1--4% in its ventral bank (subregion 7). The presylvian sulcus contained 8--12% of all PT cells in its lateral bank (subregion 2). This new cortical area is not yet considered part of 'PT cortex'. Qualitative limitations of this study are discussed.

Unmyelinated axons in the pyramidal tract of the cat.

Ultrastructural preparations revealed the presence of unmyelinated axons in the pyramidal tract (PT) of the adult cat. At the level of the medulla oblongata, unmyelinated axons constituted 8-15% of the total PT population. Axon diameters ranged from 0.05 to 0.06 micron with a mean of 0.18 micron. Although axons were distributed throughout the PT, their density was highest in the medial part.

Subcortical projections to the hippocampal formation in squirrel monkey (Saimira sciureus).

Subcortical afferents to the hippocampal formation in squirrel monkey were investigated using horseradish peroxidase as a retrograde marker. Labeled cells were found in the medial septal area, the diagonal band of Broca, anterior and laterodorsal thalamic nuclei, reuniens and periventricular thalamic nuclei, lateral hypothalamus, supramamillary nucleus, and the dorsal and superior central midbrain raphe nuclei. These results in a primate confirm previous findings in rats and cats with the exception of the noradrenergic cell groups, where the interpretation of retrograde label was hampered by high levels of endogenous pigment.

Gynecological surgical outcomes among asymptomatic human immunodeficiency virus-infected women and uninfected control subjects.

To determine the effect of asymptomatic human immunodeficiency virus (HIV) infection on the risk of complications and outcomes in women undergoing gynecological surgical procedures, retrospective analysis was performed of 62 asymptomatic HIV-infected women who underwent gynecological procedures. One hundred forty seronegative women who had similar procedures during the same time period served as controls. Procedures included tubal sterilization, hysterectomy, and diagnostic laparotomy. The following variables were compared: length of hospital stay, age, blood loss, white blood cell count, hemoglobin, and hematocrit. Laboratory parameters were compared pre-and postoperatively, as well as between the study and control groups. Race and parity were similar in both groups. HIV-infected women were younger (mean: 25 years versus 31 years) than controls. Length of hospital stay was similar. Blood loss was higher in the HIV-infected group than controls. (318 cc versus 122 cc) Differences in white blood cell counts, hematocrits, and febrile morbidity were insignificant. Asymptomatic HIV infection has minimal effect on the outcome of elective gynecologic surgery. The younger age of the HIV-infected women reflects the demographics of HIV infection and sterilization reflects the desire to prevent perinatal transmission.