Epitope mapping of a protective monoclonal antibody against Pneumocystis carinii with shared reactivity to Streptococcus pneumoniae surface antigen PspA

Pneumocystis carinii is an opportunistic fungal pathogen that causes pneumonia in the immunocompromised host. A protective monoclonal antibody (MAb) termed 4F11 generated against mouse-derived P. carinii was shown by indirect immunofluorescence assay (IFA) to bind surface antigens of P. carinii derived from multiple host species, including humans. We have identified multiple epitopes recognized by MAb 4F11 in two recombinant mouse P. carinii antigens. The epitopes mapped have similar proline content and positive charge distribution. The consensus 8-mer epitope recognized by MAb 4F11 is K/RPA/RPK/QPA/TP. Immune sera raised against intact mouse P. carinii recognized native antigens affinity purified with MAb 4F11 and a recombinant antigen reactive with MAb 4F11. Database searches for short, nearly exact matches to the mapped MAb 4F11 epitopes identified a bacterial surface antigen, Streptococcus pneumoniae PspA, with a similar proline-rich region. In an IFA, MAb 4F11 detected antigens on the S. pneumoniae surface, and Western blotting identified a protein in S. pneumoniae lysates consistent with the M(r) of PspA. A fragment of the S. pneumoniae PspA gene was cloned and sequenced, and the deduced amino acid sequence contained a region with strong similarity to the MAb 4F11 epitopes identified in P. carinii. The PspA recombinant polypeptide was recognized by MAb 4F11 in a Western blot. The ability of MAb 4F11 to recognize similar proline-rich epitopes may explain its ability to recognize P. carinii derived from multiple hosts and will permit testing of the epitopes recognized by this antibody in immunization against P. carinii.     

Salt intake by normotensive and spontaneously hypertensive rats: two-bottle and lick rate analyses

Spontaneously hypertensive rats (SHR) overconsume NaCl compared to the normotensive Wistar Kyoto rat (WKY) rat. In the present experiment, two-bottle preference for NaCl (0.01, 0.03, 0.1, 0.3, 0.5, 1.0, 3.0 M) and lick rate analyses were used to identify the possible mechanisms that underlie the intake of NaCl by male SHR. Two-bottle preference and absolute NaCl intake by SHR were greater than that of WKY rats. When NaCl intake was calculated on the basis of body weight, SHR consumed more NaCl per 100 g body weight than did WKY. Also, during the one-bottle test, SHR consumed more 0.1 and 0.3 M NaCl per 100 g body weight than did WKY. The increased intake of NaCl by SHR was most evident for 0.3 M NaCl. Intake is determined by the initial rate of licking and the decline in lick rate over time. Nonlinear regression analysis of lick rate showed that the initial lick rates (licks/min) were similar for male WKY and SHR. Lick rate declined more rapidly when WKY rats drank 0.3 M than when they drank 0.1 M NaCl, a result consistent with the role of negative feedback in controlling the decay in lick rate. This concentration-dependent change in lick rate was not seen in SHR. Also, SHR lick rate for 0.1 and 0.3 M NaCl decelerated more slowly than that of WKY rats. The increased intake of hypertonic NaCl by SHR was due to a decrease in the decline in lick rate, suggesting that SHR are less responsive to ingestion-contingent negative feedback.     

The effect of ambient temperature cycles upon circadian running and drinking activity in male and female laboratory rats

The effect of a cycle of warm and cool ambient temperature (Ta) upon the free-running circadian running and drinking rhythms of male and female laboratory rats was investigated. Rats free-running in constant darkness and constant cool Ta (21 degrees C +/- 2 degrees C) were exposed to a 12:12 cycle of high (34 degrees C +/- 2 degrees C) and cool (21 degrees C +/- 2 degrees C) Ta. Three male rats and one female rat entrained to the Ta cycle. Ten of 12 male and 9 of 11 female rats exhibited post-Ta cycle phases not predictable from pre-Ta cycle phases. Most rats exhibited positive and negative masking of activity during the Ta cycle. Activity periods shortened for all rats during the Ta cycle, and male free-running periods lengthened upon cessation of the Ta cycle to values significantly greater than precycle periods. It was concluded that Ta acts as a weak zeitgeber in laboratory rats and has greater effects on males compared to females.     

Long-term immunogenicity and efficacy of hepatitis B vaccine in homosexual men

To study the duration of antibody persistence and protection provided by the hepatitis B vaccine, we followed 773 homosexual men for five years after completion of vaccination. Among the 635 participants in whom antibody levels above 9.9 sample ratio units (SRU) developed after vaccination, 15 percent lost antibody altogether, and in another 27 percent, antibody levels declined below 10 SRU within five years. The extent of the maximal antibody response strongly predicted the persistence of protective antibody. Hepatitis B infection occurred in 55 men; 8 of these infections were clinically important (characterized by the presence of the hepatitis B surface antigen and elevation of liver-enzyme levels), and two of the patients became hepatitis B virus carriers. The long-term risk of hepatitis B infection was inversely related to the maximal antibody response to vaccine. Most severe infections occurred among those who responded poorly or had no response to the vaccination. The risk of late infection with hepatitis B in those with an initially adequate vaccine response increased markedly when antibody levels decreased below 10 SRU, but only 1 of 34 late infections resulted in viremia and liver inflammation. A second series of vaccinations induced a moderate antibody response in 50 percent of the subjects who initially had no response or a poor response; however, the persistence of antibody was poor. Both antibody loss and the risk of severe disease should be considered when booster-dose strategies for the hepatitis B vaccine are being designed.     

The prospects for and pathways toward a vaccine for AIDS

This article reviews prospects for a vaccine for acquired immunodeficiency syndrome (AIDS), considered the only certain barrier to further spread of the disease. The development of an effective vaccine against another retrovirus, feline leukemia virus, suggests the possibility that a retrovirus vaccine for humans might work. However, vaccine production depends on the ability to manufacture large quantities of a safe antigen that stimulates protective immunity when it is injected into humans. There are also many nonscientific problems that must be solved, including the reluctance of the private sector to invest in such a project. If it is found that the genetic variation of human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) is limited and predictable, vaccine-induced antibodies directed toward its structural proteins, especially envelope proteins, could protect against subsequent infection. Because retroviruses have a predilection for recombination and have been associated with malignant diseases, a live attenuated retrovirus vaccine is unlikely. A killed-virus vaccine should be more acceptable, but there are problems with selecting a cell line for producing the virus, choosing methods for purifying the viral antigens, and freeing them from viral nucleic acid. Cells derived from human tumors may be necessary to support the large-scale production of immunogenic antigens. Perhaps the best option at present is recombinant DNA technology. The advantage of antigen-production systems is that the infective virus cannot be produced because only a small portion of the viral genome is incorporated. Decisions must be made about which recombinant system to use (bacterial, yeast, or mammalian cells) and which antigen or antigens should be incorporated into the vaccine. Once expressed envelope proteins are available in adequate quantity and purity for testing, an evaluation pathway that will document safety and efficacy while minimizing the time for approval needs to be established.     

An improved systolic-diastolic pulse separator

An instrument using analogue integrated circuits for the separation of the systolic and diastolic levels of an arterial blood-pressure waveform is described. The use of the instrument in tracking the widely varying blood-pressure signals recorded from hypertensive dogs is shown. Details of the circuitry employed and the method of construction are given. An appendix describes the useful ‘ideal-diod’ circuit.     

Characterization of the Carbohydrate Moiety of Intestinal Mucin-Type Sialoglycoprotein Receptors for the K88ac Fimbrial Adhesin of Escherichia coli

We have previously identified two mucin-type sialoglycoproteins from porcine intestinal epithelial cells with approximate molecular masses of 210 (intestinal mucin-type glycoprotein IMTGP-1) and 240 kDa (IMTGP-2) as receptors for the K88ab and K88ac fimbrial adhesins of Escherichia coli. These receptors are detected in intestinal brush border membrane preparations from pigs with adhesive phenotypes but not from pigs with nonadhesive phenotypes and are postulated to be important determinants of the susceptibility of pigs to K88ab⁺ and K88ac⁺ enterotoxigenic E. coli infections. Using exoglycosidase digestion studies, we have now determined that β-linked galactose is an important component in the recognition of IMTGP-1 and IMTGP-2 by the K88ac adhesin. In addition, we observed a differential distribution of the K88ac adhesin binding activity of IMTGP-1 and IMTGP-2 along the crypt-villus axis, suggesting that receptor activity is dependent on the maturation state of the intestinal epithelial cells. Brush borders from immature intestinal epithelial cells possessed the highest concentrations of IMTGP-1 and IMTGP-2 receptor activity, with a progressive decrease in receptor activity as the cells mature. To characterize the differences in the carbohydrate moieties of IMTGP-1 and IMTGP-2, we developed a procedure for purifying the receptors, using phenol extraction followed by serial lectin affinity chromatography. Carbohydrate compositional analysis of the purified receptors indicated that the carbohydrate moieties of IMTGP-1 and IMTGP-2 consist of both N- and O-glycans containing galactose, glucose, sialic acid, mannose, N-acetylgalactosamine, N-acetylglucosamine, and fucose. The major difference between the two receptors is that IMTGP-2 contains a higher percentage of monosaccharides (mannose and glucose) commonly found in N-glycans.     

An evaluation of signs in ocular myasthenia gravis and correlation with acetylcholine receptor antibodies

Fifteen patients with ocular myasthenia gravis were examined in detail for 21 different signs, and tested for acetylcholine receptor antibodies. The major signs of ocular myasthenia gravis included ptosis, disorders of ocular rotations, weakness of eyelid closure, "pseudosupranuclear" signs and the lid twitch sign. Acetylcholine receptor antibodies were found in eight of the 15 patients. One hundred and four normal, non-myasthenic patients were also examined for the lid twitch response, and the relationship between the lid twitch of ocular myasthenia gravis and that found in normal subjects is discussed.     

Surgical treatment of cardiac pheochromocytomas

The development at our institution of the radiopharmaceutical 131-I-metaiodobenzylguanidine has permitted for the first time scintigraphic localization of pheochromocytomas. By the use of this scan in combination with contrast-enhanced computed tomography, intrapericardial pheochromocytomas have been demonstrated in eight patients at our hospital during the past 2 years. Four of these patients have been operated upon by us, and each was found to have a pheochromocytoma arising from the heart (left atrium in three and interventricular groove at the aortic root in one). While in one patient it was possible to "shell" the tumor away from the left atrial wall without cardiopulmonary bypass, in the remaining patients, bypass and cardioplegia were required to resect the pheochromocytomas without inducing life-threatening intraoperative hypertension and cardiac arrhythmias. One patient required coronary artery reconstruction and two, excision of the posterior left atrial wall with pericardial replacement. One of these latter two patients died intraoperatively of uncontrollable hemorrhage. The three remaining patients are well and normotensive after more than 1 year of follow-up. Cardiac pheochromocytomas should not be approached as typical posterior mediastinal tumors, or as they are in the abdomen, with the expectation that they will "shell away" from contiguous structures. Cardiopulmonary bypass should be available, and resection of involved myocardium may be necessary for complete removal.     

T-antigen expression by peanut agglutinin staining relates to mucosal dysplasia in ulcerative colitis

Staining of 326 rectal mucosal biopsies from ulcerative colitis patients with peanut agglutinin (PNA), which binds to the T-blood group antigen and has been claimed to reflect a cancer-associated mucin alteration, showed highly significant direct associations with mucosal dysplasia (P less than 0.001), disease activity (P less than 0.001), and subsequent development of rectal cancer in a smaller series of patients (P = 0.005). Staining for normal colonic mucin by the Dolichos biflorus (DBA) lectin related significantly and inversely to dysplasia. Intense normal colon mucin staining by DBA related significantly (P less than 0.025) to long disease duration and to subsequent development of cancer (P = 0.02). The latter association is based on a small number of patients only and is not considered conclusive evidence, but may provide a link with goblet-cell hyperplasia. The authors conclude that although T-antigen expression relates to dysplasia, the findings of "false" positive and negative rates of 22 and 33 percent respectively, make it unlikely that staining of biopsy sections for the T-antigen by peanut agglutinin will contribute materially to routine assessment for dysplasia and cancer risk prediction in patients with ulcerative colitis.