Fabrication of coupled graphene-nanotube quantum devices.

We report on the fabrication and characterization of all-carbon hybrid quantum devices based on graphene and single-walled carbon nanotubes. We discuss both carbon nanotube quantum dot devices with graphene charge detectors and nanotube quantum dots with graphene leads. The devices are fabricated by chemical vapor deposition growth of carbon nanotubes and subsequent structuring of mechanically exfoliated graphene. We study the detection of individual charging events in the carbon nanotube quantum dot by a nearby graphene nanoribbon and show that they lead to changes of up to 20% of the conductance maxima in the graphene nanoribbon, acting as a well performing charge detector. Moreover, we discuss an electrically coupled graphene-nanotube junction, which exhibits a tunneling barrier with tunneling rates in the low GHz regime. This allows us to observe Coulomb blockade on a carbon nanotube quantum dot with graphene source and drain leads.

Electronic excited states in bilayer graphene double quantum dots.

We report tunneling spectroscopy experiments on a bilayer graphene double quantum dot device that can be tuned by all-graphene lateral gates. The diameter of the two quantum dots are around 50 nm and the constrictions acting as tunneling barriers are 30 nm in width. The double quantum dot features additional energies on the order of 20 meV. Charge stability diagrams allow us to study the tunable interdot coupling energy as well as the spectrum of the electronic excited states on a number of individual triple points over a large energy range. The obtained constant level spacing of 1.75 meV over a wide energy range is in good agreement with the expected single-particle energy spacing in bilayer graphene quantum dots. Finally, we investigate the evolution of the electronic excited states in a parallel magnetic field.

The impact of pharmacogenomic factors on acute persistent rejection in adult lung transplant patients.

Persistent rejection in the face of treatment and multiple episodes of rejection are associated with the development of chronic rejection and graft loss in solid organ transplantation. The factors that create an environment for rejection that persists in the face of treatment are as yet not understood. The objective of this study was to evaluate the risk factors, including human multidrug resistance gene (MDR1), cytochrome P4503A5 (CYP3A5) and cytokine gene polymorphisms, associated with acute persistent rejection (APR) in lung transplant patients. One hundred and twenty-five adult lung transplant patients were studied. MDR1 G2677T, C3435T and CYP3A5 polymorphisms were assessed by direct sequencing of the polymorphic region in patient DNA. Cytokine genotyping for five cytokines was performed using the polymerase chain reaction-sequence specific primers (PCR-SSP) technique. Multivariate regression analysis was used to identify the predictors of acute persistent rejection. The dependent variable was the presence or absence of acute persistent rejection based on lung biopsies during the first postoperative year. The independent variables were MDR1 G2677T and C3435T, CYP4503A5 and cytokine polymorphisms, survival status, age, gender, survival days and HLA mismatches. The MDR1 C3435T polymorphism and age were independently associated with acute persistent rejection (p = 0.025, odds ratio = 0.29, 95% CI 0.1-0.86 and p = 0.016, odds ratio = 0.94, 95% CI 0.89-0.98, respectively). For the MDR1 C3435T polymorphism, 72% of patients with the C allele had acute persistent rejection in comparison to 52% for TT patients (p = 0.04). For age, a significant difference was found between the nonrejection group and the rejection group (mean+/-S.D. 52.1+/-11.2 vs. 44.4+/-12.3, p = 0.01). This is the first report of the association of a drug disposition genotype with drug-resistant acute rejection in organ transplant patients. The major predictor of acute persistent rejection in the first postoperative year for lung transplant patients was the MDR1 C3435T genotype. This association could be due to drug resistance, altered drug disposition or other immunologic effects associated with P-glycoprotein (P-gp) function. Future prospective treatment algorithms should be developed that will incorporate the knowledge of gene polymorphisms into treatment regimens to improve the outcome following lung transplantation.

The deficient migration in vitro of lung macrophages from newborn rats normalizes after three days of maturation in situ.

Because there are functional deficiencies in neonatal phagocytes, we investigated age-dependent differences in the directed migration of rat pulmonary macrophages (PM). Directed migration of PMs from newborn (less than 24 h old) rats towards partially purified rat C5a was lower than that of PMs from adults, but reached adult levels by the third post-natal day. Calculation of the dose of C5a resulting in a half-maximal migratory response (ED-50) revealed that the ED-50s for all age groups studied were similar. These results suggest that responding PMs at all times after birth have comparable sensitivity to C5a, but that a lower percentage of newborn PMs migrate toward this factor because of defects in either signal transduction or cell motility.

Modulation of endotoxin-induced neutrophil alveolitis by captopril and by hyperoxia.

We compared survival and the intensity of bronchoalveolar inflammation reflected by lung lavage after the intraperitoneal injection of endotoxin from Escherichia coli serotype 055B5 in rats breathing air and those breathing 60% oxygen for six days after endotoxin injection. Survival following 7.5 mg/kg of endotoxin was comparable in air-breathing rats (50%) and in oxygen-breathing rats (63%). Endotoxin caused a dose-dependent increase in the recovery of polymorphonuclear leukocytes from the lung. Oxygen breathing reduced the percentage of neutrophils recovered by lavage 24 hr after endotoxin from 17% to 9% after 2.5 mg/kg of endotoxin and from 34% to 12% after 7.5 mg/kg of endotoxin. The absolute number of neutrophils recovered was also significantly decreased in oxygen-breathing rats. The activity of pulmonary angiotensin-converting enzyme (ACE) has been reported to be affected by oxygen tension, and ACE degrades bradykinin, a proinflammatory mediator. Therefore, we questioned whether the salutary effect of increased inspired oxygen tension on the magnitude of neutrophil influx into the airspaces could be related to changes in ACE activity. We found that after 48 hr of peroral pretreatment of the rats with captopril, a specific ACE inhibitor, there was increased recovery of neutrophils by lavage 24 hr after injection of endotoxin in air-breathing rats. Captopril pretreatment also increased the chemotactic activity of bronchoalveolar lavage fluid (BALF). There was no concomitant alteration in the accumulation of 125I albumin in the lung following captopril pretreatment either in endotoxin-treated rats or in controls. Thus, breathing 60% oxygen decreased the accumulation of neotrophils in airspaces after intraperitoneal endotoxin injection and pharmacologic inhibition of ACE had the opposite effect. Alterations in the activity of pulmonary angiotensin-converting enzyme related to alveolar oxygen tension is a potential speculative mechanism for modulation of alveolar inflammation by the inspired oxygen concentration in this model.

Shift in subfractions of rat alveolar macrophages in vivo during endotoxin-induced alveolitis.

To elucidate changes in alveolar macrophages that accompany sepsis-induced lung injury, this study analyzed the subfractions of alveolar macrophages (AM) recovered by lung lavage during the onset of endotoxin-induced acute neutrophilic alveolar inflammation in the rat model. Centrifugation on continuous self-generated density gradients of Percoll was used to fractionate AM into subpopulations between density limits 1.012 and 1.130. Two-thirds of AM recovered from pathogen-free control rats (group C) were in a fraction with a density range of 1.058-1.078 ["normal" density fraction, (ND)]. Only 6% were located in a very low density (VLD) fraction 1.037-1.048. Neutrophils accounted for less than 1% of recovered cells and usually were found in the fraction with density range of 1.079-1.130. By contrast, if rats underwent lung lavage 15 hours after the administration of endotoxin (group E), only 38% of macrophages were recovered in the "normal" density fraction, whereas 26% of the AM recovered were in the VLD fraction. This shift in the relative sizes of the density based subpopulations coincided with the onset of acute bronchoalveolar inflammation as indicated by the recovery of neutrophils by bronchoalveolar lavage (PMN = 7 X 10(4) in C, vs. 9.4 X 10(5) in E, p less than .001). The macrophages on the low density subfractions showed functional impairment: they were less viable in culture and migrated poorly in response to endotoxin-activated serum compared to macrophages in the "normal" density fraction from the endotoxin-treated animals. The rapid emergence of the low density population after endotoxin could represent an influx of new cells, but more likely indicates that injury to or previous activation of resident macrophages has caused their density to decrease. We speculate that the emergence of a population of AM in airspaces with low density and impaired function could weaken pulmonary host defence following endotoxemia.

Abnormal ventilatory chemosensitivity and congenital myopathy.

Abnormal ventilatory function is common in patients with neuromuscular disorders. This report describes depressed ventilatory response to both hypoxia and hypercapnia, as well as arterial oxygen desaturation during sleep in a family with congenital myopathy. The index patient also had an abnormal ventilatory response to metabolic acid loading. There was clinical evidence of proximal muscle weakness, and a muscle biopsy specimen was consistent with myopathy. The reduction in ventilatory drive, however, could not be explained solely by ventilatory muscle weakness. This report describes a distinct familial syndrome of congenital myopathy and abnormal ventilatory response to hypercapnia and hypoxia. All affected family members had unique facial features, proximal muscle weakness, and impaired ventilatory responses. The combination of impaired ventilatory drive and reduced ventilatory muscle strength leaves patients particularly vulnerable, and heightened awareness of this association is important in the treatment of these patients.

Cavitary pulmonary sarcoidosis.

A 25-year-old woman was initially seen with hilar adenopathy and bilateral cavitary lesions. No cause for these roentgenographic abnormalities other than sarcoidosis could be determined. In contrast to what is usually found in acute sarcoidosis, the bronchoalveolar lavage fluid from this patient contained a normal number of lymphocytes and an increased number of multinucleated giant cells. These findings raise the possibility that patients with acute sarcoidosis and cavitation have an atypical form of this disease. Even though cavitation in acute sarcoidosis is rare, this disorder must be considered in making the differential diagnosis of cavitary lung disease.

The epidemiology of parainfluenza virus infection in lung transplant recipients.

Human parainfluenza virus (HPIV) is a common cause of seasonal respiratory tract infections. However, little is known about the clinical presentation and impact of HPIV infections in lung transplant recipients. We reviewed HPIV infections at the University of Pittsburgh Medical Center. From January 1990 through May 2000, 32 cases of HPIV infection were identified. HPIV infection was found in 24 lung transplant recipients (75%), all of whom were included in the study group. Diagnosis was established at a median of 2.1 years after transplantation (range, 0.6-5 years). Presenting symptoms included cough (17 patients), shortness of breath (16), and temperature elevation (4). Respiratory failure occurred in 5 patients (21%). The HPIV serotypes were HPIV-1 (7 patients), HPIV-2 (2), and HPIV-3 (15 [63%]). Twenty-two patients underwent transbronchial biopsy, and 18 (82%) showed signs of acute allograft rejection. Seven patients (32%) subsequently were found to have bronchiolitis obliterans.

Does histologic acute rejection in lung allografts predict the development of bronchiolitis obliterans?

Clinical acute lung rejection (AR) occurs in lung allografts usually within 50 days after transplantation. While perivascular infiltrates characterize AR, with moderate-to-severe acute rejection small airway injury occurs. We investigated the significance of small airway injury in AR and its relationship to the development of bronchiolitis obliterans (OB) in 11 recipients of combined heart-lung or double-lung allografts. In general, the intensity and persistence of early acute rejection episodes associated with injury to bronchioles correlated with the development of histologic bronchiolitis obliterans. Early AR may "prime" lymphocytes for subsequent respiratory epithelial injury and airway fibrosis late in the postoperative period.

Use of aerosolized colistin sodium in cystic fibrosis patients awaiting lung transplantation.

BACKGROUND: In patients with cystic fibrosis (CF) who are awaiting lung transplant, prolonged exposure to systemic antibiotics has frequently led to airway colonization with resistant isolates of Pseudomonas. This resistance limits the arsenal of effective antimicrobials available for infections after the initiation of immunosuppression and has been considered a theoretical deterrent to lung transplantation. METHODS: Twenty CF transplant candidates with "pan-resistant" Pseudomonas received maintenance antibiotic therapy with aerosolized colistin sodium (75 mg b.i.d.), and intravenous antibiotics were eliminated. Ten other CF candidates did not use colistin sodium. Sputum cultures and antibiotic sensitivities were followed every 3-6 weeks. RESULTS: All 20 candidates (100%) who used aerosolized colistin sodium became colonized with sensitive isolates of Pseudomonas in an average of 45.1+/-20.2 days. In contrast, only 3 of 10 CF transplant candidates (30%) who did not use colistin sodium later became colonized with sensitive isolates. The mean time to spontaneous emergence of sensitive organisms was 144.6+/-48.0 days in candidates who did not use colistin sodium and was significantly longer than in the candidates who used colistin sodium (P=0.007). The occurrence of redeveloping sensitive isolates of Pseudomonas was significantly greater in the candidates who used colistin sodium (P<0.05). Of the candidates who used colistin sodium, six have been transplanted at our institution. In five of these six recipients (83.3%) bacterial cultures taken from the explanted lungs continued to demonstrate sensitive organisms. CONCLUSION: Aerosolized colistin sodium may be a useful therapy to promote emergence of sensitive microbes in CF candidates with pan-resistant isolates of Pseudomonas.

Similarity of pulmonary rejection patterns among heart-lung and double-lung transplant recipients.

The transbronchial biopsy and clinical courses of 9 double-lung and 1 single-lung recipients surviving greater than 10 days were analyzed and compared to those of 15 heart-lung transplants performed during the same time period. Of these, 8 isolated lung (LT) and 11 heart-lung transplant (HLT) recipients survived greater than 50 days and were at risk of developing obliterative bronchiolitis believed to be a form of chronic rejection. Cyclosporine-based immunosuppression, in combination with azathioprine and steroids, was used for 22 of 25 patients. Two double-lung recipients and 1 heart-lung patient received FK506 as the sole immunosuppressive agent; 90% and 62% of LT, and 67% and 54% of HLT recipients developed acute and chronic rejection, respectively (P = NS). The average time to first episode of acute (30.2 days [LT] versus 21.5 days [HLT]) and chronic rejection (146 days [LT] versus 193.7 days [HLT]) was not different between groups (P = NS). Age (34.2 [LT] versus 29.1 [HLT]) and sex (M:F, 5:5 [LT] versus 5:10 [HLT]) were also not found to be discriminators. The histologic diagnosis of chronic rejection was associated with significant declines in FEV1.0 and FEF25-75 (P less than 0.02). There was only one instance of cardiac rejection among the heart-lung transplant recipients. Heart-lung and isolated lung transplant patients appear to be at similar risk for developing acute or chronic pulmonary rejection.

Proliferation of cytomegalovirus-primed lymphocytes in bronchoalveolar lavages from lung transplant patients.

Previous reports have described an association between cytomegalovirus infection and increased donor-specific alloreactivity of bronchoalveolar lavage (BAL) lymphocytes in transplanted lungs and a higher risk of bronchiolitis obliterans due to chronic rejection. We have postulated that during infection, intragraft CMV-specific lymphocytes are activated and release lymphokines that augment cellular rejection. This study deals with an analysis of CMV antigen induced proliferation of 28 BAL lymphocyte and 27 peripheral blood lymphocytes samples from 17 lung transplant patients with or without CMV infection. Kinetic studies of lymphocyte proliferation have shown that CMV infection of the lung allograft is associated with an accelerated response of BAL lymphocytes but not PBL, following in vitro exposure to CMV antigen. These findings indicate an accumulation of primed CMV-specific lymphocytes within the lung allograft during CMV infection. Evidence has also been obtained that primed CMV-specific lymphocytes may persist for months in BAL. We conclude that the CMV antigen induced proliferation assay is useful for studies of CMV infection in transplant patients.

Proteins of the respiratory tract after heart-lung transplantation.

Heart-lung transplant recipients represent a unique population who experience episodic lung injury caused by infection or rejection. We hypothesized that the proteins in the respiratory lining fluids of these patients might reflect and provide insights into the in vivo immunologic and inflammatory events that occur in the transplanted lung. Structural, inflammatory, and immune proteins were quantitated in 57 samples of BAL fluid recovered from 17 heart-lung recipients when infections, rejection, or neither was present. Protein levels were compared with those of normal subjects and between the clinical transplant groups. When neither infection nor rejection was present, levels of albumin, fibronectin, and immunoglobulins G, M, and A were all higher in the transplanted lungs as compared with the normal lungs. These findings suggest that a new steady state of these proteins is established in the transplanted lungs. When infection or rejection was present, there was a further significant increase in albumin, fibronectin, IgG, and especially C5a in the transplanted lungs. These findings suggest that at least some elements of host defense remain intact in the posttransplantation period despite the use of immunosuppressive drugs and a HLA-incompatible microenvironment. The profiles of recovered alveolar proteins did not, however, help to differentiate infection from rejection. This is disappointing because distinguishing between infection and rejection without examination of lung tissue remains an unresolved and important clinical problem. Nevertheless these data provide new insights into organ tolerance and defense of the newly transplanted lung from infection or rejection.

Proliferative responses of bronchoalveolar lavage lymphocytes from heart-lung transplant patients.

Donor-specific alloreactivity of bronchoalveolar lavage (BAL) lymphocytes was evaluated in the immunologic monitoring of lung transplant patients. The study dealt with 161 BAL performed on 28 transplant recipients. Unseparated BAL cells, separated BAL lymphocytes, and PBL were tested for donor-specific proliferative responses in 3-day primed lymphocyte testing (PLT), and for nonspecific proliferative responsiveness to exogenous IL-2. The proliferation data were analyzed for correlation with the status of the lung allograft assessed clinically, histologically, and by pulmonary function testing. Positive PLT responses of BAL lymphocytes were observed in 20 of 22 acute rejection episodes (91%) and in 24 of 35 cases (69%) when chronic rejection was diagnosed. During clinical quiescence donor-specific proliferative activity was demonstrated in only 4 of 35 cases (11%). Thus, acute rejection and chronic rejection correlated significantly (P less than 0.001) with donor-specific PLT reactivity of BAL lymphocytes. Though significant association with rejection was observed for the alloreactivity of unseparated BAL cells and PBL, the sensitivity of the PLT test with these cells was significantly lower than that with BAL lymphocytes. Similarly, the IL-2 proliferative activity of BAL lymphocytes was significantly increased during acute and chronic rejection. However, this test had lower sensitivity and specificity than did the donor-specific PLT. These findings suggest the usefulness of the donor-specific PLT of BAL lymphocytes as a reliable method for monitoring pulmonary rejection.

HLA-class II antigen expression in human heart-lung allografts.

Long-term survival in heart-lung transplantation has ben hindered by the development of bronchiolitis obliterans (OB), which is believed to be a manifestation of chronic rejection of the lung. Since HLA-class II antigens are involved in the rejection response, the distribution of the class II products HLA-DR, HLA-DQ, and HLA-DP were studied in normal lung, and in transplanted lung with and without OB, utilizing frozen-section immunohistochemical techniques. All three allelic products are usually expressed on the epithelial, endothelial, and mesenchymal components of the lung. Sequential transbronchial biopsies from 4 recipients before and concurrent with the diagnosis of OB were stained with serial dilutions of monoclonal antibodies to assess the level of expression of the above class II products. Increased levels of HLA-DR and HLA-DP antigens may be seen on the bronchial and bronchiolar epithelium during OB, but the changes are subtle and complicated by many other variables. Additional studies are needed to confirm these preliminary results.

Cytomegalovirus serologic status and postoperative infection correlated with risk of developing chronic rejection after pulmonary transplantation.

Twenty-seven patients received pulmonary transplants during the period since we began routine use of cytomegalovirus-seronegative blood products for CMV-seronegative recipients. Preoperative serologic status of the recipient and the occurrence of cytomegalovirus infection in the postoperative period were correlated with development of obliterative bronchiolitis (OB) as diagnosed by transbronchial biopsy (TBB). Patients included 20 heart-lung and 7 double-lung recipients. OB occurred in 18 of 27 patients. All 3 CMV seronegative recipients receiving lungs from a seropositive donor and 9 of 10 CMV recipients seropositive at the time of transplantation developed OB compared with only 6 of 14 CMV seronegative patients receiving seronegative grafts (P = 0.018). CMV infection occurred in 10/27 patients, of whom 5 were asymptomatic; 90% of these patients developed OB. Donor-specific alloreactivity, based on primed lymphocyte testing (PLT) of bronchoalveolar lavage cells was found at the time of diagnosis of OB in 23 of 27 patients. A positive PLT was significantly associated with the presence of OB (P = 0.017). We conclude that preoperative seropositive status for CMV, grafting of organs from seropositive donors, and postoperative CMV infection are significant risk factors for developing OB. That OB is, in part, an immunologically mediated form of injury and represents chronic rejection is supported by the presence of donor-specific alloreactivity in BAL lymphocytes from all recipients with OB.

Interleukin 6 and interferon-gamma gene expression in lung transplant recipients with refractory acute cellular rejection: implications for monitoring and inhibition by treatment with aerosolized cyclosporine.

BACKGROUND: The purpose of this study was to correlate cytokine gene expression from bronchoalveolar lavage (BAL) cells and peripheral blood lymphocytes (PBL) with graft histology in recipients with persistent acute rejection treated with aerosolized cyclosporine (ACsA). METHODS: We measured mRNA for interleukin (IL) 6, interferon (IFN)-gamma, and IL-10 in recipients (1) without rejection (n=13), (2) with acute rejection that responded to pulsed methylprednisolone (n=7), and (3) with "refractory" acute rejection that failed to respond to conventional immunosuppression (n=17). In the latter group, ACsA was initiated. RESULTS: BAL cell IL-6 and IFN-gamma were highest in recipients with refractory rejection compared with recipients with steroid-responsive rejection and recipients with no rejection. Improvement in rejection histology occurred in 15 of 17 recipients who were treated with ACsA. IL-6 and IFN-gamma mRNA levels from BAL cells decreased during treatment with ACsA (median IL-6:actin ratio: before treatment, 0.40 vs. after treatment, 0.003, P=0.001; IFN-gamma:actin ratio: before treatment, 0.32 vs. after treatment, 0.04, P=0.001). PBL IL-6 and IFN-gamma mRNA expression also decreased during ACsA treatment after 180 days. Expression of IL-10 mRNA from BAL and PBL did not change during ACsA treatment (0.0 vs. 0.03 and 0.0 vs. 0.02, respectively). CONCLUSIONS: IL-6 and IFN-gamma mRNA expression from BAL cells was highest in those recipients with refractory histologic acute rejection. ACsA was associated with decreased IFN-gamma and IL-6 gene expression in BAL cells and PBL.

Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 KD protein.

Human lung lavage proteins were fractionated by centrifugation and molecular sieving. An antiserum to the post-albumin fraction of the soluble proteins reacted with a 10 KD protein and this protein was isolated by conventional chromatography. The protein, which has a pI of 4.8, consists of two 5 KD polypeptides and is rich in glutamic acid, leucine, serine, and aspartic acid amino acids. The protein does not bind to concanavalin A, pancreatic elastase, leukocyte elastase, or trypsin, and lacks anti-protease activity. It constitutes about 0.15% of the soluble proteins in lung lavage. Antibodies to the 10 KD protein specifically and exclusively stain Clara cells in human, dog, and rat. Staining of granules of Clara cells was prominent in the distal bronchioles; however, the non-ciliated cells of respiratory bronchioles did not stain for the 10 KD protein. This 10 KD protein appears in fetal lungs at 21 weeks of gestation, and was present in about 10% of the primary pulmonary adenocarcinomas. As a specific marker for Clara cells, this protein could be useful in the study of development, regulation of secretion, and pathobiology of these cells.

Lymphocyte enumeration: a comparison between a modified avidin-biotin-immunoperoxidase system and flow cytometry.

The reliability and sensitivity of an indirect avidin-biotin-peroxidase (ABC) procedure for enumerating lymphocyte subpopulations was compared to flow cytometry (FC) employing direct immunofluorescence. Lymphocytes were enumerated by two different methods. For counting method I, which is the method of conventional FC, the number of immunostained lymphocytes was compared to the total number of lymphocytes present. The ABC procedure by method I detected a greater proportion of immunostained lymphocytes for all subsets tested than did FC. By counting method II, where the number of immunostained lymphocytes is compared to the total number of cells present, the ABC analysis still detected more total T cells than FC but the results for the two analyses were similar for T helper and T suppressor cells. Thus, the ABC technique appears to be a valid method for enumerating T lymphocyte subsets. Furthermore, as compared to FC, it offers the advantages of reduced cost, simplicity of understanding and performance, need for fewer cells, and a permanent record of lymphocyte staining. For these reasons, we feel that the ABC technique will enjoy widespread application for the identification of lymphocyte membrane antigens.