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Hypergammaglobulinemia and defective humoral immunity are hallmarks of HIV-1 infection. Naive B cells have been recently suggested as the major source of hypergammaglobulinemia in chronic viral infections. We recently reported that HIV-1–infected patients carry low levels of memory B cells. Here we studied whether defects in the naive and memory B cells in HIV-1–infected patients translated into hypergammaglobulinemia and defective humoral immunity against specific antigens. Naive B cells from HIV-1–infected patients exhibited abnormal expression of the activation/differentiation markers CD70 and leukocyte-associated Ig-like receptor (LAIR-1). Activated naive B cells from patients showed a significant increase in the intracellular immunoglobulin G (IgG) content ex vivo and this activated phenotype correlated to hypergammaglobulinemia and to the ability of naive B cells from patients to secrete IgG in vitro. We analyzed the levels of antibodies to tetanus toxoid, measles, and HIV-1 in relation to memory B cells and observed a significant reduction of antigen-specific antibodies in patients with low-memory B lymphocytes. Nevertheless, hypergammaglobulinemia and levels of polyspecific self-reactive antibodies were comparable in patients with normal and low memory B cells. We conclude that reduction of memory B lymphocytes in HIV-1 infection correlates with defective humoral immunity and that hyperactivated naive B cells may represent the source of abnormal IgG production in HIV-1 infection. Our results may be relevant to the design of HIV-1 therapeutical vaccines and to the clinical management of HIV-1–infected patients.

ABSTRACTThe pertussis toxin (PT) promoter region is a frequently used target for DNA-based diagnosis of pertussis and parapertussis infections. The reported polymorphism in this region has also allowed discrimination of species in mixtures with severalBordetellaspecies by their specific PCR amplicon restriction patterns. In the present study, we investigated the degree of polymorphism in order to confirm the reliability of the assay. Five different sequence types of the amplified 239- or 249-bp region were found among the 33 Bordetella pertussis, B. parapertussis, and B. bronchisepticaAmerican Type Culture Collection reference strains and patient isolates analyzed. According to the sequences that were obtained and according to the PT promoter sequences already available in the databases, restriction enzyme analysis with TaqI, BglI, andHaeII, which gave four different patterns, can be performed to reliably identify B. pertussis, B. parapertussis, and B. bronchiseptica.

The regulatory role of leukotrienes (LT) on human myelopoiesis was investigated. Mononuclear bone marrow cells from 31 healthy donors were cultivated in the presence of suboptimal concentrations of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) for 10 days in semisolid agar. The addition of LTC4or LTB4to the cultures dose-dependently stimulated myeloid stem cell proliferation. Maximal effects were observed at 10−8mol/L, at which LTC4induced a 91 % ± 23% (mean ± SEM; P= .004) and LTB4a 73% ± 22% (P= .008) increase in colony formation. In contrast, addition of the LTB4isomer 5(S), 12(S)-diHETEdid not affect the growth. LTD4exerted a weak potentiating effect on progenitor proliferation (17% ±7% growth stimulation at 10−10mol/L; P= .034), whereas LTE4was without consistent effect. Furthermore, LTC4-induced stimulation of colony formation was insensitive to the LTD4antagonist ICI 198615. The dual lipoxygenase and prostaglandin endoperoxide synthase inhibitor CL42A potently suppressed the proliferation of myeloid colonies, a suppression that could be reversed by parallel addition of LTB4or LTC4. The results suggest that both LTB4and LTC4possess strong and specific synergistic stimulatory effects on GM-CSF–induced human myeloid progenitor cell growth.

Diarrheal episodes with enterotoxigenic Escherichia coli (ETEC) were prospectively monitored during the first 2 years of life in a cohort of 235 infants from Leon, Nicaragua. ETEC was an etiological finding in 38% (310 of 808) of diarrheal episodes and in 19% (277 of 1,472) of samples taken as asymptomatic controls at defined age intervals (P = <0.0001). The majority of diarrheal episodes (80%) occurred before 12 months of age. The major ETEC type was characterized by colonization factor CFA I and elaboration of both heat-labile enterotoxin and heat-stable enterotoxin (ST). The proportion of E. coli strains with CFA I was significantly higher in cases with diarrhea (P = 0.002). The second most prevalent type showed putative colonization factor PCFO166 and production of ST. The prevalence of PCFO166 was approximately 20%, higher than reported before. Children with a first CFA I episode contracted a second ETEC CFA I infection 24% of the time, compared with 46% for ETEC strains of any subtype. Most of the ETEC episodes were of moderate severity, and only 5% (15 of 310) were characterized as severe. In conclusion, our results give valuable information for the planning of intervention studies using ETEC vaccines.

A nested PCR, using a 239-bp sequence in the pertussis toxin promoter region, was developed and evaluated. The assay differentiates Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica by restriction enzyme analysis of the amplified fragments. The diagnostic performance of the PCR was evaluated in a Swedish pertussis vaccine efficacy trial which took place from 1992 to 1995, including study children and household members and using culture and serology for laboratory confirmation of suspected cases. In total 2,421 nasopharyngeal aspirates were analyzed. The total diagnostic sensitivity for B. pertussis was 90.2% (194 of 215). During the study period samples were processed with and without the cation-exchange resin Chelex. The PCR diagnostic sensitivity for B. pertussis among the Chelex-treated aspirates was 94.9% (75 of 79), and that for B. pertussis among 124 aspirates in a consecutive non-Chelex-treated material was 89.5% (111 of 124). After Chelex treatment of the 13 PCR-negative samples, an additional six became PCR positive, giving a final sensitivity of 94.3%. In addition, PCR was positive for B. pertussis with 57 of 1,744 samples negative by culture but with available serological data. The specificity of PCR with these samples was supported by a significant increase in antibody levels between acute and convalescent sera in 45 cases and by epidemiological or clinical data in all but two of the remaining cases. PCR was also positive for B. pertussis with 26 of 415 aspirates from episodes lacking serology. The diagnostic sensitivity of PCR for B. parapertussis was 74.0% (37 of 50). There were an additional seven culture-negative B. parapertussis PCR findings, six from cases with significant antibody increases against filamentous hemagglutinin only and one from a case lacking serology. There were no samples positive for B. bronchiseptica. In conclusion, PCR detection of B. pertussis and/or B. parapertussis enabled us to identify 90 positive nasopharyngeal aspirates, in addition to the 262 culture-positive samples (an increase of 34%). Relating these cases to serology and clinical data indicated a PCR specificity approaching 100%.

In the present study, novel solid-phase methods were used for both sample preparation and PCR detection of Bordetella pertussis. The sample preparation was performed by immunomagnetic separation with paramagnetic beads coated with polyclonal antibodies directed toward the surface antigens of the bacteria. The precoated immunobeads were directly used on nasopharyngeal aspirates to capture the bacteria on the solid support and were subsequently transferred to the PCR tube with no further manipulations. The region encompassing the pertussis toxin promoter was analyzed to allow direct discrimination between the three major Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica). The resulting amplicons were captured on a second magnetic solid phase, allowing detection and restriction analysis of the target sequence. A colorimetric detection system based on a DNA binding fusion protein enabled the use of standardized enzyme-linked immunosorbent format tests both for the detection of Bordetella spp. and for species evaluation. When the optimized system was evaluated on 55 clinical aspirate samples, 21 of 22 (95%) culture-positive samples were positive by the system that we developed. In addition, two samples were positive by the PCR-based assay, while the culture assay was negative. The implications of these results are discussed.

1. In a group of blood donors the mean serum iron concentration, and plasma clearance of iron did not differ significantly from normal means whereas the mean TIBC was increased. No statistically significant correlation existed between these values and the amount of blood donated in the year preceding the study. 2. The iron absorption was significantly correlated with the amount of blood donated during the year preceding the study, and the mean absorption value was significantly higher than in normal controls. 3. Compared to healthy men higher absorption values were likewise found in a group of healthy young women, without pathologic menstruations, and with essentially normal serum iron and TIBC values. 4. In order to discover how the intestine is instructed to increase iron absorption the correlations were studied between iron absorption and hemoglobin, serum iron, TIBC and plasma clearance of iron. No significant correlations were found. It is suggested that absorption is not primarily regulated by these factors. The finding of increased absorption in persons with essentially normal serum iron, TIBC and iron clearance supports this suggestion. 5. To determine whether the high absorption observed in women was a sign of iron deficiency, absorption was again studied after combined oral and parenteral iron treatment. It decreased in each case, except one where absorption remained unchanged. 6. Parenteral treatment alone, increased the serum iron but no statistically significant difference was found in TIBC, plasma clearance of iron, or iron absorption. Absorption after parenteral treatment was correlated mainly with pretreatment absorption. 7. After oral treatment on the other hand, absorption decreased significantly. No substantial changes were noted in serum iron or plasma clearance of iron, while TIBC unexpectedly increased. The findings support the view that no change in serum iron, TIBC or the plasma iron clearance is required to bring about a decrease in iron absorption.

1. Since previous studies could not demonstrate that any of several general plasma factors played a major role in intestinal iron absorption, local intestinal factors were examined in 240 iron absorption studies on 150 healthy subjects. 2. When the iron dose was increased 40 times, from 0.25 to 10 mg. the percentage absorption was halved. 3. Trebling the quantity of food (bread) in the intestine did not significantly decrease absorption. 4. Ascorbic acid in the intestinal lumen trebled the absorption even of ferrous iron. A stable pharmaceutical combination of iron and ascorbic acid was tested. 5. Sifted flour did not seem to inhibit the absorption of ferrous iron, but coarse ground flour did. When fat was added, no further decrease in absorption was found although iron soaps may be formed. 6. A further decrease in absorption was found after a complete meal. 7. When fine grain reduced iron was used to enrich flour—this is done in all Swedish flour—absorption was 50 per cent lower, and when a coarser grain reduced iron was used 85 per cent lower, than when ferrous sulfate was used for enrichment. 8. When oral iron treatment was given to persons with high iron absorption, absorption was decreased to normal.

Results are presented which indicate that in guinea pigs cyanocobalamin undergoes conversion to hydroxocobalamin, in vivo and in vitro, at the approximate rate of 0.1-0.4 mµg./day/Gm. of liver. In boiled liver, no conversion was found. The radioactivity excreted in human urine the first 12 hours after a therapeutic cyanocobalamin dose, on the other, is still cyanocobalamin. It is suggested that demonstrated metabolic differences between therapeutically used cyanocobalamin and hydroxocobalamin are explained, in part, by the time needed to convert cyanocobalamin to hydroxocobalamin.

A technic for the study of radioiron absorption and loss is described employing an NaI (T1) crystal-detector whole body counter and 1-10 µc. Fe59 in 250 µg. elemental iron. Changes in whole body Fe59 activity during the first few hours and the next 90-100 days after oral ingestion are described and their significance discussed. Normal absorption with this technic ranges from 5.7-24.7 per cent of the administered tracer. In 14 patients with polycythemia vera, 12 previously phlebotomized and 2 with a recent history of gastrointestinal hemorrhage, iron deficiency as evidenced by increased iron absorption (20.6 per cent-96.9 per cent) correlates well with the extent of preceding phlebotomy, and relatively well with the plasma iron at the time of study. Although other parameters reflect iron deficiency, none correlate well with the absorption of radioiron. Next to increased iron absorption, depletion of iron stores in the marrow seems to be the earliest evidence of iron deficiency. Iron absorption and erythrocyte incorporation of radioiron was also studied in several other hematologic disorders, including four heavily menstruating women, three cases of aplastic anemia, and a small number of other conditions. The findings are described and discussed. Radioiron loss in three normal patients was 0.110 per cent, 0.110 per cent, and 0.182 per cent daily, and in two patients with aplastic anemia 0.103 per cent and 0.173 per cent daily, defining the normal range of tracer loss over days 20-100. Radioiron loss in the polycythemics ranged from 0-0.044 per cent daily. An unusual case of pyridoxine-responsive anemia with increased absorption of radioiron (69.1 per cent), but no red cell incorporation, lost only 0.026 per cent/day. Some problems in the interpretation of such data are discussed. The results demonstrate the effectiveness of the technic of whole body counting in the study of various aspects of iron metabolism.