Exhaled breath condensate pH assays are not influenced by oral ammonia.

BACKGROUND: Measurement of pH in exhaled breath condensate (EBC) is robust and simple. Acidic source fluid (airway lining fluid) traps bases while volatilising acids, leading to EBC acidification in many lung diseases. Lower airway ammonia is one determinant of airway lining fluid pH, raising the concern that addition of the base ammonia by contamination from the mouth might confound EBC pH assays. METHODS: Three discrete methods were used to limit oral ammonia contamination of EBC collections: endotracheal intubation, oral rinsing, and -40 degrees C condenser temperatures. Separately, ammonia was removed from collected EBC samples by lyophilisation and resuspension. Intraweek and intraday variability of ammonia concentration was determined in 76 subjects, and ammonia and pH from a further 235 samples were graphically compared. Ammonia was assayed spectrophotometrically and pH was assessed after deaeration. RESULTS: Data from 1091 samples are presented. Ammonia was reduced in EBC by all methods. Endotracheal intubation decreased EBC ammonia from a mean (SD) of 619 (124) microM to 80 (24) microM (p<0.001, n=32). Oral rinsing before collection also led to a decline in EBC ammonia from 573 (307) microM to 224 (80) microM (p=0.016, n=7). The colder the condensation temperature used, the less ammonia was trapped in the EBC. Lyophilisation removed 99.4 (1.9)% of ammonia. Most importantly, the pH of EBC never decreased after removal of ammonia by any of these methods. Intraweek and intraday coefficients of variation for ammonia were 64 (27)% and 60 (32)%, which is substantially more variable than EBC pH assays. CONCLUSIONS: Although ammonia and pH appear to correlate in EBC, the oral ammonia concentration is not an important determinant of EBC pH. No precautions need to be taken to exclude oral ammonia when EBC pH is of interest. The low pH and low ammonia found in EBC from patients with lung diseases appear to be independent effects of volatile compounds arising from the airway.

Exhaled breath condensate pH is a robust and reproducible assay of airway acidity.

Exhaled breath condensate (EBC) pH is low in several lung diseases and it normalises with therapy. The current study examined factors relevant to EBC pH monitoring. Intraday and intraweek variability were studied in 76 subjects. The pH of EBC collected orally and from isolated lower airways was compared in an additional 32 subjects. Effects of ventilatory pattern (hyperventilation/hypoventilation), airway obstruction after methacholine, temperature (-44 to +13 degrees C) and duration of collection (2-7 min), and duration of sample storage (up to 2 yrs) were examined. All samples were collected with a disposable condensing device, and de-aerated with argon until pH measurement stabilised. Mean EBC pH (n=76 subjects, total samples=741) was 7.7+/-0.49 (mean+/-SD). Mean intraweek and intraday coefficients of variation were 4.5% and 3.5%. Control of EBC pH appears to be at the level of the lower airway. Temperature of collection, duration of collection and storage, acute airway obstruction, subject age, saliva pH, and profound hyperventilation and hypoventilation had no effect on EBC pH. The current authors conclude that in health, exhaled breath condensate pH is slightly alkaline, held in a narrow range, and is controlled by lower airway source fluid. Measurement of exhaled breath condensate pH is a simple, robust, reproducible and relevant marker of disease.

Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression.

Effects of inflammatory pain states on functional and molecular properties of the rat blood-brain barrier (BBB) were investigated. Inflammation was produced by subcutaneous injection of formalin, lambda-carrageenan, or complete Freund's adjuvant (CFA) into the right hind paw. In situ perfusion and Western blot analyses were performed to assess BBB integrity after inflammatory insult. In situ brain perfusion determined that peripheral inflammation significantly increased the uptake of sucrose into the cerebral hemispheres. Capillary depletion and cerebral blood flow analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Western blot analyses showed altered tight junctional protein expression during peripheral inflammation. Occludin significantly decreased in the lambda-carrageenan- and CFA-treated groups. Zonula occluden-1 expression was significantly increased in all pain models. Claudin-1 protein expression was present at the BBB and remained unchanged during inflammation. Actin expression was significantly increased in the lambda-carrageenan- and CFA-treated groups. We have shown that inflammatory-mediated pain alters both the functional and molecular properties of the BBB. Inflammatory-induced changes may significantly alter delivery of therapeutic agents to the brain, thus affecting dosing regimens during chronic pain.

Effect of reduced flow on blood-brain barrier transport systems.

Pathological states (i.e. stroke, cardiac arrest) can lead to reduced blood flow to the brain potentially altering blood-brain barrier (BBB) permeability and regulatory transport functions. BBB disruption leads to increased cerebrovascular permeability, an important factor in the development of ischemic brain injury and edema formation. In this study, reduced flow was investigated to determine the effects on cerebral blood flow (CBF), pressure, basal BBB permeability, and transport of insulin and K+ across the BBB. Anesthetized adult female Sprague-Dawley rats were measured at normal flow (3.1 ml min(-1)), half flow (1.5 ml min(-1)), and quarter flow (0.75 ml min(-1)), using bilateral in situ brain perfusion for 20 min followed by capillary depletion analysis. Reduction in perfusion flow rates demonstrated a modest reduction in CBF (1.27-1.56 ml min(-1) g(-1)), a decrease in pressure, and no significant effect on basal BBB permeability indicating that autoregulation remained functional. In contrast, there was a concomittant decrease in BBB transport of both insulin and K+ with reduced flow. At half and quarter flow, insulin transport was significantly reduced (R(Br)%=17.2 and R(Br)%=16.2, respectively) from control (R(Br)%=30.4). Additionally, a significant reduction in [86Rb+] was observed at quarter flow (R(Br)%=2.5) as compared to control (R(Br)%=4.8) suggesting an alteration in ion homeostasis as a result of low flow. This investigation suggests that although autoregulation maintains CBF, BBB transport mechanisms were significantly compromised in states of reduced flow. These flow alterations may have a significant impact on brain homeostasis in pathological states.

Laparoscopic cholecystectomy as an outpatient procedure.

BACKGROUND: Laparoscopic cholecystectomy is still done mainly on an inpatient basis at hospitals or on an outpatient basis at ambulatory care departments inside hospitals. STUDY DESIGN: We reviewed 213 cases in which outpatient laparoscopic cholecystectomy was done at an ambulatory surgical center not associated with a hospital physically or administratively. Patients were selected solely on the basis of medical history and physical examination results. Patients received general anesthesia as is typical for outpatient procedures. Narcotic use was minimized to prevent postoperative nausea. The procedure did not include intraoperative cholangiography. RESULTS: Laparoscopic cholecystectomy took 1 to 2 hours in three quarters of patients. Rate of conversion to open cholecystectomy was 2.8% (6 of 213 patients). The mean recovery period was 6.6 hours, and 97% of patients were discharged on the same day (ie, were treated as outpatients). We identified no cases of retained common duct stone. Wound complications included mainly seroma, wound seepage, and wound infection; 18% of these complications were seen at trocar sites. No major complications were seen. CONCLUSIONS: Elective outpatient laparoscopic cholecystectomy can be done safely with low morbidity, high patient acceptance, and same-day discharge in > 95% of cases.

Enkephalin analog prodrugs: assessment of in vitro conversion, enzyme cleavage characterization and blood-brain barrier permeability.

To improve the blood-brain barrier penetration of the delta-opioid receptor peptides [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Pen2, L-Cys5]enkephalin (DPLCE), various prodrug forms were synthesized to increase lipophilicity and drug delivery to the brain. The aims of this study were 3-fold, 1) to assess the metabolic conversion of various DPDPE and DPLCE prodrugs in vitro using mouse brain homogenate and mouse serum, 2)to characterize the proteolytic enzymes responsible for cleaving prodrugs to the parent compounds using select peptidase inhibitors and 3)to assess the blood-brain barrier permeability of prodrugs, compared with their parent compounds, using the in vitro bovine brain microvessel endothelial cell culture model. The prodrugs with carboxyl-terminal phenylalanine residues (DPDPE-Phe and DPLCE-Phe) had significantly longer metabolic conversion times in both mouse serum and brain homogenates than did the prodrugs with amino-terminal phenylalanine residues. Inhibition of leucine aminopeptidase with bestatin in the serum increased the conversion time of Phe0-DPDPE from 6.8 min to 92.2 min. Inhibition of aminopeptidase M with amastatin in the brain homogenate increased the conversion time of Phe0-DPDPE from 3.9 min to > 450 min. The long half-life of DPLCE-Arg-Pro-Ala in serum (317 min) vs. brain (9.2 min) can be explained by the high levels of the degradative endopeptidase 24.15 (EC 3.4.24.15) in the central nervous system but not in plasma. The data also showed that, for specific prodrugs of DPDPE such as Phe0-DPDPE and DPDPE-Arg-Gly, the prodrug shows a significant improvement in permeability, compared with the parent compound. Therefore, these data provide evidence that prodrugs or prodrug-enzyme inhibitor combinations may optimize the delivery of peptide and/or protein drugs to the central nervous system.

In vitro stability of some reduced peptide bond pseudopeptide analogues of dynorphin A.

Eight analogues of DYN A(1-11)-NH2 incorporating the nonhydrolyzable psi [CH2-NH] peptide bond surrogate were tested for their in vitro enzymatic stability in mouse brain homogenates. Results show that the Leu(5)-Arg6 and to a lesser extent the Arg(7)-Ile8 and Ile(8)-Arg9 peptide bonds are the more susceptible to enzymatic cleavage in the native peptide. (Leu5 psi[CH(2)-NH]Arg6)DYN A(1-11)-NH2 exhibits an almost complete resistance to enzymatic cleavage with a half-life greater than 500 min in brain, compared to 42 min for the standard peptide, DYN A(1-11)-NH2.

In vitro predictors of therapeutic response in melanoma patients receiving tumor-infiltrating lymphocytes and interleukin-2.

PURPOSE: To correlate in vitro characteristics of tumor-infiltrating lymphocytes (TIL) with clinical response to TIL immunotherapy in patients with metastatic melanoma. PATIENTS AND METHODS: Forty-one melanoma patients undergoing 43 separate treatment courses with TIL and interleukin-2 (IL-2) from December 1990 through November 1992 were studied prospectively. Multiple patient and treatment characteristics were evaluated for response correlates. In addition, TIL were assayed within 7 days of infusion for characteristics such as doubling time, cell-surface phenotype, autologous tumor lysis in 4-hour chromium-51 release assays, and cytokine secretion following autologous tumor stimulation. RESULTS: Nine patients experienced complete or partial tumor regressions. Clinical parameters such as age, sex, sites of disease, performance status, and prior therapies were similar in responders and nonresponders. Treatment variables such as the cumulative IL-2 dose and concomitant administration of cyclophosphamide or interferon (IFN)-alpha were not predictive of response, although responders received 33% more TIL. However, statistically significant differences in favor of clinical response were noted for extranodal source of TIL (v lymph node), shorter culture duration (mean, 38 v 47 days), shorter TIL doubling time (2.6 v 3.7 days), greater autologous tumor lysis by TIL (30% v 15%; effector-to-target [E:T], 40:1), and secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) by TIL following autologous tumor stimulation (six of nine responders v eight of 32 nonresponders). CONCLUSION: The associations of TIL lysis of autologous tumor and younger TIL age with clinical response observed in this study are supportive of previous reports, and these findings will be useful in designing future clinical trials. The new observation correlating GM-CSF secretion by TIL with clinical response is interesting and needs further substantiation.

Inhibition of the macrophage-induced oxidation of low density lipoprotein by interferon-gamma.

The regulation of the macrophage-induced oxidation of low density lipoprotein (LDL) by cytokines was investigated. As an initial source of cytokines, medium from an activated type 2 helper T-cell clone was tested. This cell-free supernatant inhibited the subsequent oxidation of LDL by mouse peritoneal macrophages. The inhibition was concentration- and time-dependent as measured by changes in thiobarbituric acid (TBA) reactive substances. In addition, there were decreases in conjugated diene formation as well as the generation of LDL particles with an increased net negative charge that were recognized by the scavenger receptor. The inhibition was not due to a decrease in cell viability or to nonspecific antioxidant activity, as assessed by measuring phagocytic activity and metal ion-induced oxidation of LDL, respectively. Using antibodies that inactivate specific cytokines, the role of select individual cytokines in this inhibition was investigated. Addition of antibodies against interleukin-3 (IL-3), granulocyte/macrophage-colony stimulating factor (GM-CSF), or tumor necrosis factor alpha (TNF alpha) to the media had little or no effect on the ability of the cytokines to affect oxidation by macrophages, whereas anti-interferon-gamma (IFN-gamma) antibodies completely reversed the inhibition induced by the T-cell supernatant. A role for this cytokine was confirmed using recombinant IFN-gamma. A concentration-dependent inhibition was produced with a maximum inhibition to 24% of control cells, whereas smooth muscle cell-dependent LDL oxidation was not affected. To examine the cellular basis for the inhibition, the effect of IFN-gamma on oxidant activities (O2- production, lipoxygenase activity, and thiol production) were measured. IFN-gamma at concentrations that maximally inhibit LDL oxidation stimulated the phorbol myristate acetate (PMA)-induced production of O2- 1.4-times greater than control cells after one hour. Similarly, thiol production was increased 29% by IFN-gamma pretreatment. In contrast, macrophage lipoxygenase was inhibited approximately 21%. Based on these in vitro findings, the potential regulation of macrophage LDL oxidation by IFN-gamma in vivo was also investigated. Macrophages from Toxoplasma gondii-infected mice have been shown previously to be activated in situ by an IFN-gamma-dependent mechanism. These were tested for their ability to oxidize LDL. Macrophages from these mice oxidized LDL to a much lesser extent than cells from age-matched control mice, demonstrating that the ability of macrophages to oxidize lipoprotein may also be susceptible to regulation possibly also by IFN-gamma in vivo. Together these studies demonstrate that the cell-mediated oxidation of LDL can be regulated by cytokines, specifically IFN-gamma. This mode of regulation may play a role in regulating this process in the developing atherosclerotic lesion.

Hemodynamic effects of the administration of tumor-infiltrating lymphocytes to cancer patients.

Tumor-infiltrating lymphocytes (TILs) can mediate tumor regression in selected patients with advanced cancer. To study some of the physiological changes associated with TIL administration, hemodynamic effects were measured while 2 x 10(10) to 20 x 10(10) TILs (mean 10 +/- 1 x 10(10)) were infused into 22 patients. In 10 patients, the first bag of TILs was administered without any interleukin-2 (IL-2) in the infusion bag; subsequent bags in the same patients and all bags in the next 12 patients contained IL-2 in low concentrations (300,000 IU). Two hours following infusion (as compared with baseline), patients developed tachycardia (110 +/- 3.3 vs. 76 +/- 3.5 beats/min; p < 0.001), increased cardiac index (4.9 +/- 0.2 vs. 3.2 +/- 0.13 L/min/m2; p < 0.001), decreased systemic vascular resistance (677 +/- 37 vs. 1185 +/- 63 dyn/s/cm5; p < 0.001), and increased pulmonary artery diastolic pressure (15.9 +/- 1.4 vs. 10.6 +/- 1.1 mm Hg; p = 0.002). No significant changes in systemic blood pressure were noted. Analysis of data obtained in the 10 patients after infusion of the first bag of TILs (4.5 +/- 0.4 x 10(10)) without IL-2 present in the infusate revealed similar, though less severe, changes. No significant correlation was noted between in vitro production of tumor necrosis factor-alpha, interferon-gamma, and granulocyte-macrophage colony-stimulating factor by TILs and hemodynamic effects when administered to patients. These results indicate that TIL infusion can cause hemodynamic changes similar to those previously reported in patients undergoing IL-2 therapy.

Specific release of cytokines by lymphocytes infiltrating human melanomas in response to shared melanoma antigens.

Lysis of autologous and human leucocyte antigen (HLA)-matched allogeneic melanomas by cultured human tumor-infiltrating leukocytes (TIL) suggests that shared melanoma antigens (Ag) exist and are recognized by TIL in the context of self major histocompatibility complex (MHC) molecules. We have recently shown that cytokine release by TIL is another indicator of the specific interaction with autologous tumor. To determine if recognition of shared melanoma Ag can also induce cytokine release, seven melanoma TIL, which lysed autologous tumor, were co-cultured with autologous tumor or with 7-12 HLA-matched or unmatched melanoma stimulators for 6-24 h. Supernatants were collected and assayed by ELISA for the presence of granulocyte/macrophage colony stimulating factor (GM-CSF), interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-6. Among five of six melanoma TIL for which autologous tumor was available, autologous melanoma cells stimulated specific release of at least one of three cytokines: GM-CSF, IFN-gamma, and TNF-alpha. Neither IL-4 nor IL-6 secretion by three TIL cultures tested was enhanced upon contact with tumor. For six of seven TIL cultures, HLA-matched allogeneic melanomas also stimulated significant cytokine release; HLA-A1, -A2, -A24, -B8, and -Cw7 were identified as possible restriction elements. The cytokine secretion induced by both autologous and allogeneic HLA-matched melanomas could be blocked by an anti-MHC I antibody. These data suggest that cytokines can be specifically released by TIL recognizing a shared melanoma antigen in the context of self MHC molecules.

Specific immune recognition of autologous tumor by lymphocytes infiltrating colon carcinomas: analysis by cytokine secretion.

Tumor-infiltrating lymphocytes (TIL) were grown in the presence of interleukin-2 from 19 colon carcinoma specimens, including 1 primary lesion and 18 metastatic lesions. These cultures showed a median proliferation of 606-fold (range 13-fold to 28,000-fold) over 49 culture days (range 26-76 days). By phenotype, mature cultures were 69%-99% CD3+ (mean 93%) and contained mixed populations of CD4+ and CD8+ cells (CD4 > CD8 in 10 of 19 cultures). Fresh cryopreserved colon tumors were not lysed by autologous TIL in short-term 51Cr-release assays, and were poorly lysed by lymphokine-activated killer cells. Ten TIL cultures were assayed for cytokine secretion in response to autologous and allogeneic tumors during a 6- to 24-h coincubation. Culture supernatants were tested by ELISA for the presence of granulocyte/macrophage-colony-stimulating factor, interferon gamma, and tumor necrosis factor alpha. Of 10 TIL, 4 secreted at least two of these cytokines specifically in response to autologous and/or HLA-matched fresh allogeneic colon carcinomas, but not to melanomas or HLA-unmatched colon carcinomas. Cytokine secretion was mediated by both CD4+ and CD8+ TIL, and could be inhibited by mAb directed against the appropriate class of MHC antigen. These data provide evidence for specific, MHC-restricted immune recognition of human colon carcinomas by T lymphocytes.

Recognition of shared melanoma antigens by human tumor-infiltrating lymphocytes.

We have established that melanomas express shared tumor antigens (Ags) that can be recognized by T cells if presented in the context of self-MHC molecules. Tumor-infiltrating lymphocytes (TILs) from six melanoma patients were tested for lysis of large panels of HLA-matched or unmatched targets representing a variety of tissue types. Lysis was specific for allogeneic melanomas sharing at least one HLA-A, -B, or -C Ag with TILs, and demonstrated commonly expressed tumor Ags. Similar findings were obtained when cytokine secretion by TILs was used to indicate specific Ag recognition. Transfection of the HLA-A2.1 gene into HLA-A2- melanoma lines conferred susceptibility to lysis by HLA-A2 restricted melanoma TILs, demonstrating expression of common tumor Ags among patients of diverse HLA types. These findings have important implications for developing broadly applicable cancer immunotherapies such as vaccines.

Common expression of melanoma tumor-associated antigens recognized by human tumor infiltrating lymphocytes: analysis by human lymphocyte antigen restriction.

Major histocompatibility complex (MHC) class I antigens (Ag), particularly human lymphocyte antigen (HLA)-A2, have been shown to function as restriction elements in human cytotoxic T lymphocyte recognition of tumor. This study was undertaken to determine the function of non-A2 MHC class I Ag in tumor recognition by tumor-infiltrating lymphocytes (TILs) cultured from six melanomas, and to find evidence for shared or unique tumor-associated Ag. Four predominantly CD8+ and two mixed CD4+, CD8+ population TIL cultures were tested for lysis in short-term 51Cr-release assays against a panel of targets including 29 fresh melanomas, 2 fresh sarcomas, 11 cultured melanoma lines, and 14 nonmelanoma cell lines derived from HLA-typed patients. All six melanoma TILs lysed the autologous melanoma. Two of three TILs from HLA-A2+ patients lysed allogeneic melanomas matched for HLA-A2, giving evidence for shared tumor Ag; one of these TILs also used HLA-B44 as a restriction element. The third HLA-A2+ TIL lysed autologous melanoma but not autologous Epstein-Barr virus-transformed B cells nor 14 HLA-A2 matched allogeneic melanomas, suggesting the possibility of a unique tumor Ag in this system. The three HLA-A2- TILs each lysed multiple HLA-matched melanomas, using HLA-A24, HLA-A31, and HLA-Cw7 as restriction elements. Blocking of autologous and allogeneic melanoma lysis by TILs with mAb w6/32 (anti-MHC class I) and anti-CD3, as well as cold target inhibition assays, confirmed that specific interaction of the T-cell receptor with MHC class I Ag and the relevant tumor Ag on the target cell surface is required for tumor lysis. These data provide evidence for specific recognition of shared melanoma Ag by human TILs.

Attempted scapholunate arthrodesis for chronic scapholunate dissociation.

We report a retrospective review of seven patients with chronic scapholunate dissociation treated with attempted scapholunate arthrodesis. All procedures were done between 1978 to 1983 by one surgeon. Seven patients with follow-up of nine months or greater were available for review. Follow-up for the four patients with successful operations averaged seventy-seven months. Average age was 34 years (range from 19 to 46). Only one patient demonstrated radiographic fusion. Of the remaining six patients, three had no further wrist pain. The other three operations were total failures requiring other procedures for pain relief. For all patients, flexion-extension are decreased an average of 4 degrees. Grip strength averaged 88% of the unaffected side. Lateral scapholunate angle improved from an average preoperative angle of 70 degrees to a postoperative angle of 59 degrees. We conclude that this procedure is not a predictable method to treat scapholunate dissociation.

Postoperative gastric atony after vagotomy for obstructing peptic ulcer.

From 1979 through 1984, truncal vagotomy and drainage were performed in 71 patients with symptomatic obstructing peptic ulcers, whereas proximal gastric vagotomy with or without drainage was performed in 30 patients. Seven patients (7 percent) developed prolonged early postoperative gastric atony. Six of the 71 patients (8 percent) who had truncal vagotomy had atony, whereas only 1 of the 30 patients (3 percent) with proximal gastric vagotomy had atony (p = 0.08). The atony resolved with medical management in all patients after a median of 23 days. At follow-up (median 3 years), 74 percent of patients with truncal vagotomy had an excellent or good result compared with 86 percent of those with proximal gastric vagotomy (p greater than 0.1). The conclusion was that prolonged early postoperative gastric atony occurs uncommonly after vagotomy for obstructing peptic ulcer. Preservation of antropyloric innervation by using proximal gastric vagotomy instead of truncal vagotomy may be helpful, but does not completely prevent the atony.

Evaluation of the effectiveness of BCG vaccination using the case-control method in Buenos Aires, Argentina.

A retrospective case-control study was conducted in Argentina to determine the protection conferred by BCG vaccination against tuberculosis in children under six years of age, in an area where coverage is about 55%. A total of 175 tuberculosis patients were included. Five controls selected from patients treated at the same hospital as those under study for reasons other than tuberculosis were matched to each case on the basis of age, socioeconomic origin, nutritional status and place of residence. Information on BCG vaccination status was collected by an independent examiner. Tuberculosis localizations were as follows: 152 pulmonary, pleural and/or miliary; 18 meningitis; 2 lymphadenitis; 2 osteoarticular; and 1 otic. The diagnosis was based on bacteriological and histopathological tests, computerized tomography, radiology, clinical examination, endoscopy, and proved source of infection. The protective effect of BCG among those who were vaccinated was 73.0% with 95% confidence limits of 82% and 62%. According to these results BCG vaccination given early in life is very effective in preventing tuberculosis.