Immunocytochemical analysis of cellular responses to BCG.

The study reported here was performed to find out whether changes in the number of mycobacteria in various organs of BCG-infected mice can be related to changes in the phenotype of monocytes, macrophages and lymphocytes in the blood, various tissues, and peritoneal cavity and to the formation of granulomas in the spleen, liver and lungs. The relative amounts of various antigens on the leukocytes were assessed semi-quantitatively after immunocytochemical detection of the binding of monoclonal antibodies. Granuloma formation was determined after immunocytochemical staining of cells in sections of liver and lung tissue with a monoclonal antibody against the common leukocyte antigen and in sections of the spleen with a monoclonal antibody against the Mac-2 antigen. The results showed that during the first week of infection the number of BCG in spleen, liver and lungs declined considerably. Multiplication of mycobacteria during the second week of infection was associated with decreased expression of antigen F4/80 and increased expression of Ia antigen and Mac-2 antigen by blood monocytes and macrophages. Reduction of the numbers of BCG in the spleen and liver during the third week after i.v. injection of BCG and in lungs during the fourth week of the infection was found to be correlated with the degree of granuloma formation in these organs. After intravenous injection of killed BCG no changes were observed in the phenotype of monocytes and the macrophages in spleen, liver, lungs and peritoneal cavity. These mice showed considerably less granuloma formation than BCG-infected mice. The present results indicate that live but not killed mycobacteria induce macrophage activation.     

Human smooth pursuit during transient perturbations of predictable and unpredictable target movement

Summary The predictive component of human smooth pursuit was studied by perturbing sinusoidal target motion at unpredictable instants. The disturbances consisted of either a brief period of stabilization of the target on the fovea or a replacement of the sine by a ramp displacement for half a period. To minimize the effects of a possible change of the tracking strategy by the subject the transitions were masked and only the initial part of the response to the disturbance was analyzed. After stabilization on the fovea the eye oscillation continued at the frequency of the preceding target movement for about one half-cycle, whereupon the oscillation was rapidly damped. The mean unidirectional smooth eye acceleration was 70% of the mean unidirectional target acceleration prior to the stabilization. This suggests that during pursuit of a sinusoidal target movement about 75% of the oculomotor response is generated by predictive processes. When the sine was replaced by a ramp, starting at the velocity zero-crossing, the eye accelerated away from the target for ca. 180 ms irrespective of the frequency of prior tracking. In contrast, when the ramp started at the peak velocity of the sinusoidal target motion the eye accelerated away from the target for more than a quarter period. After foveal stabilization during pursuit of a pseudorandom stimulus, the eye continued to oscillate for less than one period at approximately the highest frequency present in the stimulus. The frequency characteristics of human smooth pursuit of predictable as well as unpredictable target motion were correctly simulated by a model, which derived its predictive properties from a lead element, tuned to the current frequency of the target motion.     

Contribution of molecular typing methods and antifungal susceptibility testing to the study of a candidemia cluster in a burn care unit.

We investigated a cluster of cases of Candida septicemia diagnosed in four burn patients. Twenty clinical isolates of Candida albicans and two of Candida parapsilosis, plus eight isolates of C. albicans recovered from nurses' clothes, were analyzed by antifungal susceptibility testing and three genotyping methods (restriction fragment length polymorphism analysis with EcoRI and HinfI, arbitrarily primed PCR, and karyotyping). The high MICs of the azoles for all of the C. albicans isolates tested suggest either a natural resistance of the endogenous flora or the transmission of isolates with acquired resistance. The genotyping methods demonstrated the involvement of four different strains, cross-infections with one C. albicans strain and one C. parapsilosis strain, and identity between some of the strains from the patients and nurses. The origins of the strains remain unclear. Our results show that the use of a combination of at least two different methods such as those used in the present study is recommended for C. albicans typing.     

Sex ratio of the mutation frequencies in haemophilia A: coagulation assays and RFLP analysis.

Coagulation and RFLP data from 41 families with an isolated haemophilia A patient were used to estimate the sex ratio of mutation frequencies (nu/mu). Based on the results of coagulation assays in all the female relatives investigated, nu/mu was estimated to be 12.1 by the maximum likelihood method (95% confidence interval 3.8 to 62.5). In order to avoid the possible influence of germline mosaicism, an additional analysis was performed in which only the results in the mothers and grandmothers of an isolated patient were included. The nu/mu ratio was then estimated to be 5.2 (95% confidence interval 1.8 to 15.1). Because an estimate of nu/mu based on all available RFLP data can easily be biased in favour of males, we set up a model in which only information on the grandparental derivation of the patient's X chromosome was used, irrespective of the generation in which the mutation actually occurred. In this way nu/mu was estimated to be minimally 4. The probability of carriership for mothers of an isolated haemophilia A patient amounts to 86% with a sex ratio of 5.2. Although this would imply that 14% of the mothers are not carriers of the disease in the classical sense, they may be mosaic for the mutation and, therefore, also at risk of transmitting the mutation more than once.     

Localization of immunogenic regions on the flagellin proteins of Campylobacter jejuni 81116.

The purpose of this study was to localize antigenic regions on the flagellin protein of Campylobacter jejuni 81116. This strain has two flagellin genes, flaA and flaB, which are 95% identical; only flaA seems to be expressed in motile C. jejuni 81116 cells. Fragments of flaA and flaB were cloned in the bacterial expression vector pEX, and the expression products were incubated with flagellin-specific antibodies. Monoclonal antibodies to broadly cross-reactive epitopes recognized fragments that are located in the termini (CF16 and CF17) and in the center (CF15) of both flagellin A and B proteins. Most of the serotype-specific monoclonal antibodies (CF1, CF2, CD3, CF4, and CF13) reacted with only the center of flagellin A in an area where flagellin A and B differ in 6 amino acid residues. The epitopes in this area were further characterized by competitive binding experiments. The charge and molecular weight microheterogeneity of flagellin, as determined by two-dimensional gel electrophoresis, were unrelated to the expression of both flagellin genes or parts thereof.     

Smooth muscle differentiation in cultured human breast gland stromal cells

We analyzed in cultures from the human breast the potential of stromal cells resembling fibroblasts to undergo smooth muscle differentiation. The cellular components of the breast tissue from 10 biopsies were disaggregated by collagenase digestion and further purified by differential centrifugation into suspensions of single cells and intact blood vessels. These two fractions of stromal cells were plated in culture and their phenotypic traits analyzed within 24 hours. During this time, the blood vessel fraction gave rise to stromal cells with smooth muscle differentiation as judged immunocytochemically using monoclonal antibodies to alpha-/gamma-muscle actins, to alpha-smooth muscle actin, to type IV collagen, and to laminin. Furthermore, the cells of this fraction resembled smooth muscle cells based on 2D gel electrophoresis and immunoblotting determination of isoactin content. After 24 hours in culture, the single-cell fraction consisted of an almost pure population of cells not exhibiting smooth muscle differentiation but rather resembling fibroblasts. Maintenance of fibroblast-like cells without smooth muscle differentiation was possible for more than 14 days on chemically defined medium. These cells remained quiescent, as measured by cell quantification and immunoreactivity to the proliferation-associated antigen, Ki-67. Growth of these cells could be stimulated by adding serum at any time during the experimental period. Single-cell fractions from seven biopsies were allowed to grow exponentially in the presence of serum for up to 10 days, and the kinetics of smooth muscle differentiation were monitored immunocytochemically and biochemically. These experiments showed that alpha-smooth muscle actin synthesis was induced in 10 to 80% of the fibroblast-like cells after 4 to 11 days in culture. Both the final number of alpha-smooth muscle actin-positive cells and the onset of synthesis varied with the initial seeding density. Dose-response experiments (at constant cell density) revealed that serum exerted maximal effect at concentrations above 10%. It was therefore concluded that elements of smooth muscle differentiation may arise in non-smooth muscle stromal cells taken directly from human breast tissue.     

Ventilatory anaerobic threshold in healthy children

Summary The ventilatory anaerobic threshold (VAT) during graded exercise was defined as the oxygen uptake ( ) immediately below the exercise intensity at which pulmonary ventilation increased disproportionally relative to . Since VAT is considered to be a sensitive and noninvasive measure for evaluating cardiorespiratoy endurance performance, the purpose of the present study was to determine normal values in children. We examined 257 healthy children (140 boys and 117 girls) varying in age from 5.7 to 18.5 years, during treadmill exercise. The data were analyzed in relation to sex and age. In boys the lowest (ml · min⁻¹ · kg⁻¹) was found in the youngest age group (5–6 year). In girls, on the other hand, no significant increase occurred with age. For VAT, expressed as ml O₂ · min⁻¹ · kg⁻¹ or as a percent of a significant decrease was found in boys and girls with age. This suggests an increase in lactacid anaerobic capacity during growth. In contrast to observations in adults, only low correlations were found between and VAT (r=0.28 in boys and r=0.52 in girls), which suggests that the development of the underlying physiological mechanism does not occur at the same rate in growing children. These data provide normal values for VAT that can be used for clinical exercise testing in the pediatric age group.     

Human ocular counterroll: assessment of static and dynamic properties from electromagnetic scleral coil recordings

Summary Static and dynamic components of ocular counterroll as well as cyclorotatory optokinetic nystagmus were measured with a scleral search coil technique. Static counterroll compensated for about 10% of head roll when the head was tilted to steady positions up to 20 deg from the upright position. The dynamic component of counterroll, which occurs only while the head is moving, is much larger. It consists of smooth compensatory cyclorotation opposite to the head rotation, interrupted frequently by saccades moving in the same direction as the head. During voluntary sinusoidal head roll, cyclorotation compensated from 40% to more than 70% of the head motion. In the range 0.16 to 1.33 Hz, gain increased with frequency and with the amount of visual information. The lowest values were found in darkness. The gain increased in the presence of a visual fixation point and a further rise was induced by a structured visual pattern. Resetting saccades were made more frequently in the dark than in the light. These saccades were somewhat slower than typical horizontal saccades. Cyclorotatory optokinetic nystagmus could be induced by a patterned disk rotating around the visual axis. It was highly variable even within a same subject and had in general a very low gain (mean value about 0.03 for stimulus velocities up to 30 deg/s). It is concluded that cyclorotational slip velocity on the retina is considerably reduced by counterroll during roll of the head, although the residual cyclorotation after the head has reached a steady position is very small.     

Comparison of PCR with the routine procedure for diagnosis of tuberculosis in a population with high prevalences of tuberculosis and human immunodeficiency virus

Direct smear examination with Ziehl-Neelsen (ZN) staining for the diagnosis of tuberculosis (TB) as employed in most low-income countries is cheap and easy to use, but its low sensitivity is a major drawback. The low specificity of chest X-rays, used for the diagnosis of smear-negative TB, risks high levels of overdiagnosis. Major advances in molecular techniques, which rapidly identify mycobacterial DNA in sputa, may overcome these obstacles. In this study, the AMPLICOR PCR system was used to diagnose pulmonary TB in a developing country with high prevalences of both TB and human immunodeficiency virus (HIV). The sensitivity and specificity of this technique were compared to those of the usual diagnostic techniques. Sputum specimens were collected from 1,396 TB suspects attending the Rhodes Chest Clinic, Nairobi, Kenya. The specimens were analyzed for the presence of Mycobacterium tuberculosis by PCR; culture on Löwenstein-Jensen medium was used as the “gold standard.” All culture-positive samples were genotyped to identify the mycobacterial species. The sensitivity and specificity of PCR were 93 and 84%, respectively. HIV status did not affect the sensitivity of PCR. A total of 99.7% of the true smear-positive and 82.1% of the true smear-negative TB patients were correctly identified by PCR. PCR detected M. tuberculosis in 11.7% of the culture-negative suspects, 60% of which had one or two PCR-positive sputum specimens. Of the 490 positive cultures, 486 were identified as M. tuberculosis. The high sensitivity of Amplicor PCR merits usage in a clinical setting with high TB and HIV burdens. Thus, PCR can be considered as an alternative to ZN staining in combination with chest X-ray for diagnosis of TB; however, cost-effectiveness studies and operational studies are required to support an evidence-based decision of introducing PCR for TB control in high-burden environments.Tuberculosis (TB) is one of the most serious infectious diseases and a considerable public health problem due to its high risk of person-to-person transmission, morbidity, and mortality. Both the human immunodeficiency virus (HIV) epidemic and social deterioration have contributed to the overall increase in the Mycobacterium tuberculosis infection rate, especially in developing countries, where resources are scarce (13). In Nairobi the case detection rate increased from 78 per 100,000 in 1991 to 581 per 100,000 in 2001, with a total number of 12,963 cases.Early diagnosis followed by adequate treatment is essential to prevent both morbidity and mortality. Although the conventional technique of direct smear examination with Ziehl-Neelsen staining (ZN) is cheap and easy to perform, its low sensitivity is a major drawback. Depending on the number of specimens examined, ZN detects 30 to 60% of the culture-positive “TB suspects” (7). Furthermore, it requires sputum samples collected on consecutive days, making the procedure slow and making patient compliance with the diagnostic process difficult.New techniques are very much needed (7), and molecular amplification assays such as PCR have been shown to be promising alternatives even for developing countries (2). PCR has the potential to be a cost-effective alternative, provided the diagnosis can be determined with one sputum examination (8). If diagnosis can be established faster, and the diagnostic process becomes less cumbersome for the patient, PCR may reduce delay both in diagnosis and in the start of treatment.Depending on the “gold standard” and other methodological factors, studies show PCR sensitivities ranging from 77% to more than 95% and PCR specificities of >95% for smear-positive specimens (4, 9, 10, 12). However, sensitivities for smear-negative TB patients have been reported to be below 90% (9). Most PCR studies have been performed in industrial countries (4, 9, 10, 12) where the TB and HIV burdens are low.To investigate the performance and feasibility of PCR in an environment of TB endemicity and high prevalences of HIV and AIDS, a study was conducted in Nairobi, Kenya, comparing PCR to conventional routine diagnostic methods within a program setup. In this study, the Roche Amplicor Mycobacteria PCR test for the direct detection of M. tuberculosis was used on sputum specimens from TB suspects attending a chest clinic in Nairobi. Its performance was compared with those of the basic routine diagnostic procedures according to the national guidelines (6), including clinical findings, ZN, and chest X-rays (CXR), on smear-negative suspects. Löwenstein-Jensen (LJ) culture results were used as the gold standard.     

Tissue-specific tumor suppressor activity of retinoblastoma gene homologs p107 and p130

The retinoblastoma gene family consists of three genes: RB, p107, and p130. While loss of pRB causes retinoblastoma in humans and pituitary gland tumors in mice, tumorigenesis in other tissues may be suppressed by p107 and p130. To test this hypothesis, we have generated chimeric mice from embryonic stem cells carrying compound loss-of-function mutations in the Rb gene family. We found that Rb/p107- and Rb/p130-deficient mice were highly cancer prone. We conclude that in a variety of tissues tumor development by loss of pRB is suppressed by its homologs p107 and p130. The redundancy of the retinoblastoma proteins in vivo is reflected by the behavior of Rb-family-defective mouse embryonic fibroblasts in vitro.