Protein uptake into multivesicular bodies and storage granules in the fat body of an insect.

The fat body in Calpodes ethlius (Lepidoptera, Hesperiidae) takes up protein from the blood throughout the larval stage before pupation. Depending upon the phase of development, the protein appears in multivesicular bodies, in large storage granules, and in structures of intermediate form. There are three phases in the 8 days of the last larval stage; the first devoted to growth (molting to 66 hr), the second to synthesis for storage or export (M + 66 to M + 156 hr), and the third to preparation for pupation (M + 156 to pupation at M + 192 hr). From M + O to M + 156 and from M + 180 to M + 188 hr, protein is taken up into multivesicular bodies. Larger MVB's form a continuous series with the protein granules formed from M + 162 to M + 180 hr. Blood proteins increase in concentration and amount from M + 66 to M + 156 hr at the same time as the fat body cells have a high rate of incorporation of amino acids and a structure appropriate for protein synthesis. During granule formation, both amino acid incorporation and blood protein concentration decrease. Since foreign proteins injected into the blood appear in the granules, they are probably made mainly from sequestered blood. Protein uptake involves two stages: concentration between the cells, and ingestion in pinocytotic vesicles. The vesicles fuse to become MVB's or storage granules, depending upon their rates of growth and the addition of lytic enzymes. Since MVB's do not accumulate in the fat body and since many of them contain acid phosphatase and appear empty, they are presumed to be concerned in protein turnover.

Protein uptake in multivesicular bodies in the molt-intermolt cycle of an insect.

Plant peroxidase injected into the hemocoel is taken up in granules by almost all tissues. These granules may become multivesicular bodies or isolation bodies which later breakdown. There is most uptake and breakdown at times in the molt-intermolt cycle when cells are engaged in active syntheses.

Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma.

We have attempted to identify mRNA for IL-5 in endobronchial mucosal biopsies from asthmatics and controls, using the technique of in situ hybridization. Bronchial biopsies were obtained from 10 asthmatics and 9 nonatopic normal controls. A radio-labeled cRNA probe was prepared from an IL-5 cDNA and hybridized to permeabilized sections. These were washed extensively before processing for autoradiography. An IL-5-producing T cell clone derived from a patient with the hyper-IgE syndrome was used as a positive control. As a negative control, sections were also treated with a "sense" IL-5 probe. Specific hybridization signals for IL-5 mRNA were demonstrated within the bronchial mucosa in 6 out of the 10 asthmatic subjects. Cells exhibiting hybridization signals were located beneath the epithelial basement membrane. In contrast, there was no hybridization in the control group. No hybridization was observed with the sense probe. The six IL-5 mRNA-positive asthmatics tended to have more severe disease than the negative asthmatics, as assessed by symptoms and lung function, and showed a significant increase in the degree of infiltration of the bronchial mucosa by secreting (EG2+) eosinophils and activated (CD25+) T lymphocytes. Within the subjects who showed positive IL-5 mRNA, there was a correlation between IL-5 mRNA expression and the number of CD25+ and EG2+ cells and total eosinophil count. This study provides evidence for the cellular localization of IL-5 mRNA in the bronchial mucosa of asthmatics and supports the concept that this cytokine regulates eosinophil function in bronchial asthma.

Comparison of DNA amplification and immunofluorescence for detecting Pneumocystis carinii in patients receiving immunosuppressive therapy.

Two studies were performed to compare the sensitivity of DNA amplification with immunofluorescence for the detection of Pneumocystis carinii in asymptomatic normal and immunosuppressed subjects receiving no anti-Pneumocystis chemoprophylaxis. In the first study, immunofluorescence and silver stains were used to examine 12 induced sputa and 12 bronchoalveolar lavage specimens from 24 normal control subjects; induced sputa from 20 renal transplant recipients; and induced sputa from 11 patients with fibrosing alveolitis. All specimens were negative for P carinii using both stains, apart from one renal patient in whom 2 P carinii cysts were seen by immunofluorescence alone. In the second study, DNA amplification and immunofluorescence were used to examine induced sputa from 3 groups of 10 control, renal, and heart/lung transplant recipients. All 30 specimens were negative for P carinii by immunofluorescence. However, 3 renal and 2 heart/lung patients were positive for P carinii by DNA amplification alone. One of these patients developed P carinii pneumonia 6 weeks after sputum induction. DNA amplification is a more sensitive technique than immunofluorescence for detecting P carinii. P carinii colonization occurs in asymptomatic organ transplant recipients, but not in normal individuals.

Quantitative and qualitative comparison of DNA amplification by PCR with immunofluorescence staining for diagnosis of Pneumocystis carinii pneumonia.

AIM: To compare the results of DNA amplification by the polymerase chain reaction (PCR) with immunofluorescence staining for detecting Pneumocystis carinii in bronchoalveolar lavage specimens taken from symptomatic HIV seropositive patients with suspected P carinii pneumonia (PCP). METHODS: Bronchoalveolar lavage specimens were obtained from 28 symptomatic HIV seropositive patients. Specimens were examined for P carinii using immunofluorescence, and by DNA amplification with PCR to obtain results on gel electrophoresis (gel) and a more sensitive Southern hybridisation (blot) technique. Specimens positive by immunofluorescence and gel electrophoresis were serially diluted to a 10(-6) concentration and each dilution strength tested for P carinii using PCR to compare quantitatively immunofluorescence with PCR. RESULTS: Of the 28 specimens analysed, 18 were negative for P carinii by both immunofluorescence and PCR, two were positive only by the blot technique of PCR, four were equivocally positive and four unequivocally positive by immunofluorescence. Three of the four equivocally positive patients tested by immunofluorescence were negative for P carinii by PCR, although one was positive by PCR (blot) technique. This patient had clinically confirmed PCP. Of the four unequivocally positive patients tested by immunofluorescence, three were gel and blot positive by PCR and had PCP clinically, but one was negative by both gel and blot techniques, although the patient certainly had PCP on clinical grounds. This patient had received nine days of treatment with high dose co-trimoxazole before bronchoalveolar lavage specimens were obtained. The three specimens positive by gel and blot techniques remained gel positive down to dilutions of between 10(-4) and 10(-6). CONCLUSIONS: PCR results may become negative soon after starting treatment for PCP. Specimens should therefore be taken before, or soon after, starting treatment. PCR seems to be between 10(4) and 10(6) times more sensitive than immunofluorescence.

Serum IgM antibody and influenza A infection.

Sucrose density gradient ultracentrifugation followed by haemagglutination inhibition for demonstrating specific influenza IgM was evaluated as a means of confirming recent infection with influenza A viruses. Specific IgM antibodies were found in at least one serum obtained from 83% of patients with proven recent infection with influenza A viruses but in none of the sera from 21 individuals without evidence of infection. Influenza IgM antibodies persisted for up to 112 days after infection. The relative merits of detecting specific IgM and complement fixing antibodies for diagnostic purposes are discussed.

Detection of antibodies of the IgM class in sera of patients recently infected with influenza viruses.

Sucrose density gradient ultracentrifugation can be used to detect specific 19S antibodies of the IgM class in the sera of patients recently infected with influenza A virus, provided steps are taken to remove non-specific inhibitors of haemagglutination. The usefulness of the procedure for the diagnosis of influenza requires further evaluation.

Enteroviruses, endoscopy and infection control: an applied study.

Decontamination methods for medical equipment are based largely on bacterial studies yet enteroviruses are more resistant to disinfection than most vegetative bacteria and other viruses. To study the elimination of enteroviruses from endoscopes, poliovirus was aspirated into the suction-biopsy channels of five gastroscopes. Endoscopes were cleaned in detergent and disinfected in 2% alkaline glutaraldehyde. Contamination was measured before and after cleaning and after various periods of disinfection by irrigating the channels with viral medium and quantifying surviving virus by plaque assay. The effectiveness of glutaraldehyde against cell-free and cell-associated polio virus, dried to a surface in a protein coagulum, was also studied. Cleaning reduced virus by a mean of 4.6 log10 plaque forming units (pfu) ml-1. Samples were virus-free after 2 min disinfection. Virus dried on surfaces was inactivated in 1 min by 2% and 1% glutaraldehyde, with a reduction of > 6 log10 pfu ml-1. Thus, cleaning was effective against heavy viral contamination while glutaraldehyde rapidly inactivated poliovirus even when dried to a surface in serum.

Viral transmission and fibreoptic endoscopy.

Fibreoptic endoscopes have been responsible for outbreaks of infection with bacteria although viral transmission has been reported only once. The emergence of human immunodeficiency virus (HIV) has prompted a review of infection control practices in endoscopy units because of the theoretical possibility that HIV might be transmitted at endoscopy. Recent studies have shown that bronchoscopes and gastroscopes used on AIDS patients become contaminated with HIV genetic material although cleaning equipment in detergent removes all traces of the virus. Thorough precleaning has been shown to eliminate even high titres of HIV from endoscopes and 2% alkaline glutaraldehyde has been found to inactivate the virus rapidly even if the virus is dried in serum to a surface. These findings support the British Society of Gastroenterology recommendations for the cleaning and disinfection of endoscopic equipment and demonstrate that a uniform policy of infection control is practicable in endoscopy units.

A study of glutaraldehyde disinfection of fibreoptic bronchoscopes experimentally contaminated with Mycobacterium tuberculosis.

Mycobacteria are difficult to inactivate, and concern about the spread of tuberculosis at bronchoscopy has a major influence on infection control practices. Recommendations from the UK Department of Health are based largely on in-vitro mycobactericidal assays which do not take into account the particular conditions encountered in endoscopy units. In this applied study cleaning and disinfection methods were examined using five bronchoscopes that were heavily contaminated with a recent isolate of Mycobacterium tuberculosis in sputum. Cleaning reduced contamination by a mean 3.5 log(10) colony forming units (cfu) per ml; all bronchoscopes were free of detectable mycobacteria after 10 min in 2% alkaline glutaraldehyde (AG). It is recommended that all bronchoscopes be thoroughly pre-cleaned and disinfected in 2% AG for 20 min as part of a uniform policy of infection control.

Steroid trials in the assessment of reversibility of air flow limitation: a survey of current clinical practice of chest physicians.

To evaluate how steroid trials are currently used in the assessment of reversibility of air flow limitation, a postal questionnaire was sent to 355 consultant members of the British Thoracic Society working in England and Wales; 253 questionnaires were returned (71% response rate). Two respondents did not undertake steroid trials; of the remaining 251, 75% prescribed 30-40 mg oral prednisolone, with the commonest treatment period being 2 weeks. A high dose steroid inhaler was sometimes used as an alternative by 31% of respondents. Although 71% of respondents made lung function measurements on several occasions before starting steroids and 76% made measurements during treatment, 78% assessed patients on only one occasion at the end of the trials to ascertain its outcome. Weight, blood pressure and glycosuria were measured less frequently after the steroid treatment compared to the pre-trial period. Blood glucose and serum electrolytes were infrequently measured both before and after treatment. Wide variations exist in steroid trial regimens and current practice may neither provide definitive evidence of treatment benefit nor an adequate safeguard for patients against potential side-effects.

A comparison of several agents with two delivery systems for the prevention of airway narrowing induced by nebulised pentamidine isethionate.

Bronchial narrowing is the major side effect of inhaled nebulised pentamidine isethionate, used for the prophylaxis and treatment of Pneumocystis carinii pneumonia. Several agents and delivery systems were assessed for prophylaxis of bronchial narrowing in HIV-positive males receiving regular nebulised pentamidine isethionate. In a previous study we found the mean maximum fall in FEV1 with nebulised pentamidine alone to be 21%. FEV1 was measured before and after inhaling nebulised pentamidine, preceded by one of the following bronchodilator/immunoregulatory agents: Terbutaline metered dose inhaler (500 micrograms), nebulised salbutamol (5 mg), nebulised ipratropium bromide (500 micrograms), nebulised sodium cromoglycate (20 mg), and nedocromil sodium metered dose inhaler (4 mg). Each agent was administered once only to ten different subjects. Nebulised salbutamol gave most effective prophylaxis against bronchial narrowing induced by nebulised pentamidine (mean maximum fall in FEV1 = 5% vs. 21%, P less than 0.001). Terbutaline given by metered dose inhaler was significantly less effective than high dose terbutaline (10 mg) given by nebuliser, demonstrated in the previous study (mean maximum fall in FEV1 = 14% vs. 6%, P less than 0.05). Mean maximum falls in FEV1 for ipratropium bromide, sodium cromoglycate and nedocromil sodium were 16, 17 and 16%, respectively. High dose beta 2-agonists administered by nebuliser give more effective prophylaxis against nebulised pentamidine-induced bronchial narrowing than either lower doses given by metered dose inhaler, anticholinergics or immunoregulatory drugs.

DNA amplification by the polymerase chain reaction to detect sub-clinical Pneumocystis carinii colonization in HIV-positive and HIV-negative male homosexuals with and without respiratory symptoms.

Pneumocystis carinii colonization was studied in 90 men using the polymerase chain reaction. These comprised ten heterosexual controls; ten HIV-seronegative homosexual controls; 20 HIV-seropositive homosexuals with blood CD4 count > 400 x 10(6) l-1; 20 HIV-seropositive homosexuals with CD4 < 400 x 10(6) l-1; ten HIV-seropositive homosexuals with CD4 < 60 x 10(6) l-1 receiving PCP chemoprophylaxis; and 20 HIV-seropositive homosexuals with respiratory symptoms but without PCP. Induced sputum was obtained from all but the last group, who had bronchoalveolar lavage, and all specimens were tested for P. carinii using the polymerase chain reaction. The first four groups received no pneumocystis chemoprophylaxis, and all but the last group were asymptomatic. P. carinii colonization did not occur in the two control groups. P. carinii colonization rates were significantly different in the CD4 > 400, CD4 < 400, and CD4 < 60 groups (10%, 20%, and 40% respectively) (P < 0.025). Two patients (one each from CD4 < 400 and CD4 < 60) developed PCP 4-6 weeks after sputum induction, both had previously had high levels of P. carinii on sputum induction. Two patients from the CD4 < 400 group had high levels of P. carinii but did not develop PCP. In the symptomatic group, two subjects had low levels of P. carinii, but did not develop PCP. We have demonstrated P. carinii colonization in HIV-seropositive homosexuals in association with a low peripheral CD4 count. The polymerase chain reaction may be a useful technique for determining the need and efficacy of anti-pneumocystis chemoprophylaxis.

Diagnosis, treatment and outcome of pneumonia in the acquired immune deficiency syndrome.

Fibreoptic bronchoscopy was performed in 43 of 52 consecutive patients with opportunistic pneumonia in the acquired immune deficiency syndrome (AIDS). The 15 patients in whom a likely pathogen was not found by bronchoscopy (including the nine not having the procedure) were treated for Pneumocystis carinii pneumonia (PCP) alone and all responded. In 11 of these a diagnosis of AIDS was confirmed because of an alternative opportunistic infection within 6 months. PCP was confirmed in 38 of the 52 patients and cytomegalovirus (CMV) was isolated from 15 patients. The lower the partial pressure of arterial oxygen (PaO2) on admission the more likely was a pathogen to be found by bronchoscopy. The admission PaO2 while the patient was breathing room air was the single most reliable prognostic indicator. The mean PaO2 for survivors was 9.6 kPa and 6.7 kPa for non-survivors (P less than 0.01 Student's t-test), with 50% mortality for patients with a PaO2 of less than 8 kPa on admission. Temperature and pulse rate were sensitive indicators of response to treatment, obviating the need for frequent arterial gas measurements and chest radiography. Our findings suggest that although fibreoptic bronchoscopy contributed little to the treatment and final outcome of the infection, it identified the causative pathogen in most patients.

The use of inhaled radioisotopes for measuring the effect of sputum induction on tracheobronchial clearance rates.

Inhaled radioisotopes were employed to study the role of tracheobronchial clearance in sputum induction, a technique used to diagnose Pneumocystis carinii pneumonia in patients with acquired immune deficiency syndrome (AIDS). Seven normal nonsmoking male subjects inhaled a nebulized aerosol of technetium-labelled human serum albumen, which was cleared from the lung solely by tracheobronchial clearance. The aerosol's particle size distribution ensured both alveolar and proximal airway deposition, the site of P. carinii organisms and tracheobronchial clearance mechanisms, respectively. Pulmonary emission counts were measured for 12 continuous 5-min periods before, and immediately after, sputum induction with nebulized 3% saline. A further 10-min scan was performed at 24 h to determine the alveolar fraction of deposited aerosol. Tracheobronchial clearance rates (log10[activity]/time) were calculated after log linear regression, for the time periods before, during and after sputum induction, having corrected for isotope decay and alveolar deposition. Results were analysed by the Wilcoxon rank sum test. Tracheobronchial clearance rates increased significantly in all subjects during sputum induction, with a mean 65.5% reduction in pulmonary activity over this period. Mean clearance gradients for the three time periods before, during and after sputum induction were -1.2 x 10(-3) min-1, -15.0 x 10(-3) min-1 and 0.4 x 10(-3) min-1, respectively (P < 0.025), which probably underlies the principal mechanism for success of the technique.

Chemical inactivation of HIV on surfaces.

To assess whether alcohol and glutaraldehyde are effective disinfectants against dried HIV the virucidal effects of 70% alcohol (ethanol and industrial methylated spirit) and 1% and 2% alkaline glutaraldehyde were tested against cell associated and cell free HIV dried on to a surface. Virus stock (100 microliters) or 10,000 cultured C8166 T lymphocytes infected with HIV were dried onto sterile coverslips and immersed in 2% and 1% alkaline glutaraldehyde and 70% ethanol for 30 seconds and one, two, four, and 10 minutes, there being an additional time point of 20 minutes for cell free virus disinfected with 70% industrial methylated spirit. In addition, virus stock in neat serum was tested with 1% and 2% alkaline glutaraldehyde to see whether the fixative properties of glutaraldehyde impair its virucidal properties. Virus activity after disinfection was tested by incubating the coverslips (cell associated virus) or the coverslips and sonicated cell free virus with C8166 T lymphocytes. The lymphocytes were examined for the formation of syncytia and HIV antigens were assayed in the culture fluid. Both 2% and 1% alkaline glutaraldehyde inactivated cell free HIV within one minute; 2% alkaline glutaraldehyde also inactivated cell free virus in serum within two minutes, but a 1% solution was ineffective after 15 minutes' immersion. Cell associated HIV was inactivated by 2% alkaline glutaraldehyde within two minutes. Seventy per cent industrial methylated spirit failed to inactivate cell free and cell associated HIV within 20 and 15 minutes, respectively, and 70% ethanol did not inactivate cell free virus within 10 minutes. Seventy per cent industrial methylated spirit and ethanol are not suitable for surface disinfection of HIV. Fresh 2% solutions of alkaline glutaraldehyde are effective, but care should be taken that they are not too dilute or have not become stale when used for disinfecting HIV associated with organic matter.

Infection control revisited: dilemma facing today's bronchoscopists.

The emergence of the acquired immune deficiency syndrome has provoked a widespread review of policies for infection control. Incomplete knowledge about the human immunodeficiency virus (HIV), hepatitis B virus, and mycobacteria has until now been compensated for by adopting "overkill" precautions for patients who were thought to harbour these organisms. This policy is no longer tenable, given the difficulty in identifying infected patients. The control of infection in hospitals must instead be based on the routine use of high standards of hygiene that are adequate to contain all pathogens. Attempts by bronchoscopists to formulate such a policy have been frustrated by the lack of a suitable disinfectant and by ignorance of the susceptibility of microorganisms to cleaning and disinfection in a clinical environment.