Reduced IL-10 secretion by CD4+ T lymphocytes expressing mutant cystic fibrosis transmembrane conductance regulator (CFTR).

Expression of the CFTR protein is thought to be physiologically important only in exocrine epithelial cells. However, chronic respiratory inflammation and infection remain unexplained phenomena in disease pathogenesis. Non-transformed, antigen-responsive CD4+ T cells cloned from healthy controls and CF patients homozygous or heterozygous for the delta F508 mutation transcribed CFTR mRNA and expressed immunoreactive cytoplasmic CFTR protein. T cell clones (TCC) from controls and CF patients displayed equivalent Ca(2+)-mediated Cl- current; however, TCC from patients with CF but not controls displayed defective cAMP-mediated Cl-current. Although CF-derived TCC preserved mitogen and antigen proliferative responses and specificity to tetanus toxoid epitopes, they selectively secreted approximately 45% less IL-10 compared with control TCC after activation with concanavalin A (Con A) (624 +/- 101 versus 1564 +/- 401 pg/ml per 10(6) cells, respectively; P = 0.04) or anti-CD3/phorbol ester (5148 +/- 1634 versus 11788 +/- 2390 pg/ml; P = 0.05). This difference was independent of atopy. Secretion of interferon-gamma, IL-2, and IL-4 was comparable in CF and control TCC after both forms of activation, while IL-5 was reduced in CF TCC following anti-CD3/phorbol myristate acetate (PMA) but not after Con A. We conclude that expression of mutant CFTR in human TCC is accompanied by ion channel dysfunction characteristic of the CF phenotype, and is accompanied by a reduction in IL-10 secretion after polyclonal activation. It is possible that disruption of IL-10-mediated anti-inflammatory homeostasis may contribute to early onset sustained inflammation in CF airways.

Recognition of sleep-disordered breathing in children.

OBJECTIVE: To determine whether upper airway resistance syndrome (UARS) can be recognized and distinguished from obstructive sleep apnea syndrome (OSAS) in prepubertal children based on clinical evaluations, and, in a subgroup of the population, to compare the efficacy of esophageal pressure (Pes) monitoring to that of transcutaneous carbon dioxide pressure (tcPCO2) and expired carbon dioxide (CO2) measurements in identifying UARS in children. STUDY DESIGN: A retrospective study was performed on children, 12 years and younger, seen at our clinic since 1985. Children with diagnoses of sleep-disordered breathing were drawn from our database and sorted by age and initial symptoms. Clinical findings, based on interviews and questionnaires, an orocraniofacial scale, and nocturnal polygraphic recordings were tabulated and compared. If the results of the first polygraphic recording were inconclusive, a second night's recording was performed with the addition of Pes monitoring. In addition, simultaneous measurements of tcPCO2 and endtidal CO2 with sampling through a catheter were performed on this second night in 76 children. These 76 recordings were used as our gold standard, because they were the most comprehensive. For this group, 1848 apneic events and 7040 abnormal respiratory events were identified based on airflow, thoracoabdominal effort, and Pes recordings. We then analyzed the simultaneously measured tcPCO2 and expired CO2 levels to ascertain their ability to identify these same events. RESULTS: The first night of polygraphic recording was inconclusive enough to warrant a second recording in 316 of 411 children. Children were identified as having either UARS (n = 259), OSAS (n = 83), or other sleep disorders (n = 69). Children with small triangular chins, retroposition of the mandible, steep mandibular plane, high hard palate, long oval-shaped face, or long soft palate were highly likely to have sleep-disordered breathing of some type. If large tonsils were associated with these features, OSAS was much more frequently noted than UARS. In the 76 gold standard children, Pes, tcPCO2, and expired CO2 measurements were in agreement for 1512 of the 1848 apneas and hypopneas that were analyzed. Of the 7040 upper airway resistance events, only 2314 events were consonant in all three measures. tcPCO2 identified only 33% of the increased respiratory events identified by Pes; expired CO2 identified only 53% of the same events. CONCLUSIONS: UARS is a subtle form of sleep-disordered breathing that leads to significant clinical symptoms and day and nighttime disturbances. When clinical symptoms suggest abnormal breathing during sleep but obstructive sleep apneas are not found, physicians may, mistakenly, assume an absence of breathing-related sleep problems. Symptoms and orocraniofacial information were not useful in distinguishing UARS from OSAS but were useful in distinguishing sleep-disordered breathing (UARS and OSAS) from other sleep disorders. The analysis of esophageal pressure patterns during sleep was the most revealing of the three techniques used for recognizing abnormal breathing patterns during sleep.

Home nasal continuous positive airway pressure in infants with sleep-disordered breathing.

OBJECTIVE: To review our experience with home nasal continuous positive airway pressure (CPAP) in infants with small upper airways and abnormal breathing during sleep. STUDY DESIGN: Seventy-four infants with sleep-disordered breathing and narrow upper airways, as identified by nocturnal polygraphic recording and endoscopic evaluation, were treated at home with nasal CPAP. Infants with craniofacial anomalies and trisomy 21, and infants who had been referred to us as having had "apparent life-threatening events," made up the majority of the population. Because of the rapid growth of infants, regular follow-up visits were scheduled to adjust CPAP and mask size. RESULTS: Seventy-two infants were successfully treated at home with nasal CPAP; there were two failures. Follow-up lasted from 5 months to 12 years. Compliance was not a problem, but home nasal CPAP was prescribed only for infants who lived close to our center and whose families and pediatricians were willing to support compliance. COMMENTS: Home nasal CPAP requires careful, in-laboratory titration and regular follow-up to adjust both pressure and mask size. With the support of families and pediatricians, home nasal CPAP can be an effective treatment for infants with upper airway respiratory problems during sleep. In many cases, it can provide an interim solution, enabling physicians to plan surgery at an appropriate time and giving infants time to grow before having to undergo surgical stress.

Activation of CFTR chloride current by nitric oxide in human T lymphocytes.

Nitric oxide, which is produced by cytokine-activated mononuclear cells, is thought to play an important role in inflammation and immunity. While the function of nitric oxide as a direct cytotoxic effector molecule is well established, its function as a transducer molecule in immune cells is not. By use of whole-cell patch clamp recordings, we show that nitric oxide activates cystic fibrosis transmembrane conductance regulator CI- currents in normal human cloned T cells by a cGMP-dependent mechanism. This pathway is defective in cystic fibrosis-derived human cloned T cells. These findings not only delineate a novel transduction mechanism for nitric oxide but also support the hypothesis that an intrinsic immune defect may exist in cystic fibrosis.

Tumoricidal and immunomodulatory activities of drugs and implications for therapy of mice bearing a late stage MOPC-315 plasmacytoma.

The effectiveness of a relatively low dose of cyclophosphamide (15 mg/kg CY), melphalan (2.5 mg/kg L-PAM) or the monofunctional form of CY (150 mg/kg MoCY) for the cure of mice bearing a large primary s.c. MOPC-315 tumor and extensive metastases has been shown to be dependent on the cooperation of the drugs' tumoricidal activity with T-cell-dependent antitumor immunity, the latter facilitated by the drug's immunomodulatory activity. Here, we have compared the curative effectiveness of three additional drugs: methyl nitrosourea (MNU), hydroxyurea (OH-urea) and bis-chloroethyl nitrosourea (BCNU). Among these drugs, only a relatively low dose of BCNU (15-20 mg/kg) was effective in curing most mice (85%) bearing a large, late stage tumor. A higher dose of BCNU (40 mg/kg, LD10) was much less effective. After an optimal dose of BCNU, the proliferative capacity of the tumor cells 24 h after therapy was reduced by greater than 97%. However, viable tumorigenic cells were still present in the primary tumor and enhanced T-cell-dependent antitumor immunity was necessary for their eradication. The cured mice were resistant to tumor rechallenge. When a low curative dose of L-PAM was followed by OH-urea, the therapeutic effectiveness was not affected, but when this dose of L-PAM was followed by a high nontoxic dose of MNU (100-150 mg/kg), the therapeutic effectiveness was diminished even though MNU was highly tumoricidal (i.e. greater than 99% inhibition of proliferative activity). Thus, BCNU appears to be similar to CY, L-PAM and MoCY in its mechanism of MOPC-315 tumor eradication. The alkylating activity of CY, L-PAM, MoCY and BCNU appears to be critical for their combined tumoricidal and immunomodulatory effects. Since BCNU is the simplest of these four drugs with respect to metabolic pathway, a further study with BCNU and related constructs may shed some light on the biochemical mechanisms of their mode of action. At least one reason for the ineffectiveness of OH-urea or MNU at either low or nontoxic high doses was poor tumoricidal or immunomodulatory activity, respectively. Thus, it seems important to consider both the tumoricidal and immunomodulatory activities of drugs when developing regimens for effective chemotherapy.

Melphalan-induced enhancement of tumor cell immunostimulatory capacity as a mechanism for the appearance of potent antitumor immunity in the spleen of mice bearing a large metastatic MOPC-315 tumor.

Exposure of MOPC-315 cells from the primary tumor nodule to a low concentration (0.5 nmol/ml) of melphalan (L-phenylalanine mustard; L-PAM) rendered the tumor cells capable of bringing about the generation of a potent primary antitumor cytotoxic response. Accordingly, the level of antitumor cytotoxicity generated by normal spleen cells immunized in vitro with L-PAm-treated tumor cells was at least five-fold greater than the level generated in response to untreated tumor cells. The marked superiority of L-PAM-treated tumor cells over untreated tumor cells in bringing about the generation of antitumor cytotoxicity was evident over a wide range of responder to stimulator cell ratios. The higher level of antitumor cytotoxicity exhibited by normal spleen cells immunized with L-PAM-treated tumor cells as compared with untreated tumor cells was not merely the result of direct drug-mediated tumoricidal activity, thereby reducing the number of tumor cells present which can act as cold target cell inhibitors during the 51Cr release assay. This is apparent from the observation that the level of antitumor cytotoxicity generated in response to a given percentage of stimulator tumor cells pretreated with 0.5 nmol L-PAM/ml, a drug concentration associated with retention of 60% tumor cell proliferative capacity, is substantially greater than that generated in response to less than half that percentage of untreated stimulator tumor cells. Moreover, stimulator tumor cells exposed to a fully antiproliferative concentration of L-PAM brought about the generation of a higher level of antitumor cytotoxicity than stimulator tumor cells exposed to mitomycin C at a concentration which inhibited the proliferation of the tumor cells to the same extent as the L-PAM. A low concentration of L-PAM which was effective in rendering isolated tumor cells from the primary tumor nodule capable of bringing about the generation of antitumor cytotoxicity was also effective in inducing the appearance of potent antitumor immune potential in tumor bearer splenic cells containing metastatic tumor cells.

Melphalan-mediated potentiation of antitumor immune responsiveness of immunosuppressed spleen cells from mice bearing a large MOPC-315 tumor.

Administration of a low dose of L-PAM (0.75 mg/kg) to mice bearing a large SC MOPC-315 tumor and extensive metastases led to the development of augmented antitumor immune potential in their hitherto immunosuppressed spleen cells. Such drug-induced potentiation of antitumor immune responsiveness appeared by day 2 after chemotherapy, and it could not be further enhanced but was actually reduced by depletion of glass-adherent cells, a procedure which is effective in depleting the cells known to have inhibitory activity (i.e., macrophages and metastatic tumor cells). To establish that L-PAM can lead to selective in situ abrogation of the inhibitory effectiveness of the splenic macrophages and metastatic tumor cells, we demonstrated that incubation of immunosuppressed tumor-bearer spleen cells with a low concentration of L-PAM in vitro also resulted in augmented antitumor immune potential that could not be further augmented by depletion of glass-adherent cells. L-PAM-mediated enhancement of the antitumor immune potential of immunosuppressed tumor bearer spleen cells was due at least in part to the effects of the drug on the splenic metastatic tumor cells. Isolated tumor cells treated with a low concentration of L-PAM were not only devoid of inhibitory activity for the primary in vitro antitumor immune response by normal spleen cells, but actually manifested a strong immunostimulatory capacity. Thus, L-PAM given at a low dose enhances the development of potent antitumor immunity which brings about the eradication of a large tumorigenic load that remains after the drug has been cleared from the circulation.

Increase in the effectiveness of melphalan therapy with progression of MOPC-315 plasmacytoma tumor growth.

Following inoculation with 1 X 10(6) MOPC-315 tumor cells, a single injection of a very low dose of melphalan (L-PAM, L-phenylalanine mustard), 0.75 mg/kg, cured most of the mice bearing a day 11 large primary tumor (20 mm) and metastases, but failed to cure mice bearing a day 4 nonpalpable tumor. Treatment of mice bearing a nonpalpable tumor with the very low dose of drug compromised the ability of the mice to respond effectively to the same low dose of drug when the tumor became large (day 12). However, a nonpalpable tumor could be eradicated by treatment of tumor bearers with a low dose of L-PAM, if it was present concomitantly with a large tumor on the contralateral side. A high dose of L-PAM, 15 mg/kg, cured mice bearing either a nonpalpable or a large tumor. The eradication of the tumor induced by the high dose of L-PAM appeared to be due solely to the tumoricidal effect of the drug. On the other hand, the eradication of the tumor by the low dose of L-PAM also required the participation of antitumor immunity of the host, since subsequent injection of antithymocyte serum abrogated the curative effect of the drug in most mice. Mice cured by a high dose of L-PAM were not resistant to subsequent lethal tumor challenge. In contrast, mice cured by the low dose of L-PAM were able to reject a tumor challenge of 300 times the minimal lethal tumor dose. The results obtained with L-PAM therapy are similar to the results that we had previously reported with cyclophosphamide therapy. Thus, the timing of therapy with a low dose of drug for mice bearing a MOPC-315 tumor is critical for successful therapy. Moreover, the selection of a low dose rather than a high dose of drug to eradicate a large tumor offers the advantage that it results in long-lasting potent antitumor immunity as a consequence of the participation of host antitumor immunity in the eradication of the tumor.