Synonymous mutations make dramatic contributions to fitness when growth is limited by a weak-link enzyme.

Synonymous mutations do not alter the specified amino acid but may alter the structure or function of an mRNA in ways that impact fitness. There are few examples in the literature, however, in which the effects of synonymous mutations on microbial growth rates have been measured, and even fewer for which the underlying mechanism is understood. We evolved four populations of a strain of Salmonella enterica in which a promiscuous enzyme has been recruited to replace an essential enzyme. A previously identified point mutation increases the enzyme's ability to catalyze the newly needed reaction (required for arginine biosynthesis) but decreases its ability to catalyze its native reaction (required for proline biosynthesis). The poor performance of this enzyme limits growth rate on glucose. After 260 generations, we identified two synonymous mutations in the first six codons of the gene encoding the weak-link enzyme that increase growth rate by 41 and 67%. We introduced all possible synonymous mutations into the first six codons and found substantial effects on growth rate; one doubles growth rate, and another completely abolishes growth. Computational analyses suggest that these mutations affect either the stability of a stem-loop structure that sequesters the start codon or the accessibility of the region between the Shine-Dalgarno sequence and the start codon. Thus, these mutations would be predicted to affect translational efficiency and thereby indirectly affect mRNA stability because translating ribosomes protect mRNA from degradation. Experimental data support these hypotheses. We conclude that the effects of the synonymous mutations are due to a combination of effects on mRNA stability and translation efficiency that alter levels of the weak-link enzyme. These findings suggest that synonymous mutations can have profound effects on fitness under strong selection and that their importance in evolution may be under-appreciated.

An orthologous non-MHC locus in rats and mice is linked to CD4+ and CD8+ T-cell proportion.

CD4+ and CD8+ T cells have a central role in the immune system due to their ability to protect against infection and cancer development without targeting self. Consequently, changes in CD4+ and CD8+ T-cell homeostasis can be indicative of an array of serious illnesses, ranging from viral infections to autoimmune diseases. In addition to environmental influences, there is evidence for a genetic component regulating the proportion of CD4+ and CD8+ T cells in lymphoid organs. Indeed, identifying the genetic determinants defining the frequency of the T-cell subsets is critical as it may reveal a targetable genetic pathway to modulate CD4+ and CD8+ T-cell numbers, which could be of clinical relevance for multiple disease settings. In this study, we aim to uncover non-MHC genetic factors regulating the proportion of CD4+ and CD8+ T cells in lymphoid tissues. By investigating linkage analyses on three independent F2 cohorts, namely a rat F2 (BBDP × ACI.1U.LYP) cohort, a mouse 3A9 TCR transgenic F2 (B10.BR × NOD.H2k) cohort and a mouse F2 (C57BL/6 × FVB/N) cohort, we uncover an orthologous non-MHC locus on rat chromosome 1 and mouse chromosome 7 that is linked to T-cell proportion amongst total lymphocytes.

Investigation of hollow cylindrical metal terahertz waveguides suitable for cryogenic environments.

The field of terahertz (THz) waveguides continues to grow rapidly, with many being tailored to suit the specific demands of a particular final application. Here, we explore waveguides capable of enabling efficient and accurate power delivery within cryogenic environments (< 4 K). The performance of extruded hollow cylindrical metal waveguides made of un-annealed and annealed copper, as well as stainless steel, have been investigated for bore diameters between 1.75 - 4.6 mm, and at frequencies of 2.0, 2.85 and 3.4 THz, provided by a suitable selection of THz quantum cascade lasers. The annealed copper resulted in the lowest transmission losses, < 3 dB/m for a 4.6 mm diameter waveguide, along with 90° bending losses as low as ~2 dB for a bend radius of 15.9 mm. The observed trends in losses were subsequently analyzed and related to measured inner surface roughness parameters. These results provide a foundation for the development of a wide array of demanding low-temperature THz applications, and enabling the study of fundamental physics.

Inflammation and tuberculosis: host-directed therapies.

Tuberculosis (TB) is an airborne infectious disease that kills almost two million individuals every year. Multidrug-resistant (MDR) TB is caused by strains of Mycobacterium tuberculosis (M. tb) resistant to isoniazid and rifampin, the backbone of first-line antitubercular treatment. MDR TB affects an estimated 500,000 new patients annually. Genetic analysis of drug-resistant MDR-TB showed that airborne transmission of undetected and untreated strains played a major role in disease outbreaks. The need for new TB vaccines and faster diagnostics, as well as the development of new drugs, has recently been highlighted. The major problem in terms of current TB research and clinical demands is the increasing number of cases of extensively drug-resistant and 'treatment-refractory' TB. An emerging scenario of adjunct host-directed therapies is intended to target pulmonary TB where inflammatory processes can be deleterious and lead to immune exhaustion. 'Target-organ-saving' strategies may be warranted to prevent damage to infected tissues and achieve focused, clinically relevant and long-lasting anti-M. tb cellular immune responses. Candidates for such interventions may be biological agents or already approved drugs that can be 're-purposed' to interfere with biologically relevant cellular checkpoints. Here, we review current concepts of inflammation in TB disease and discuss candidate pathways for host-directed therapies to achieve better clinical outcomes.

Efficient coupling of double-metal terahertz quantum cascade lasers to flexible dielectric-lined hollow metallic waveguides.

The growth in terahertz frequency applications utilising the quantum cascade laser is hampered by a lack of targeted power delivery solutions over large distances (>100 mm). Here we demonstrate the efficient coupling of double-metal quantum cascade lasers into flexible polystyrene lined hollow metallic waveguides via the use of a hollow copper waveguide integrated into the laser mounting block. Our approach exhibits low divergence, Gaussian-like emission, which is robust to misalignment error, at distances > 550 mm, with a coupling efficiency from the hollow copper waveguide into the flexible waveguide > 90%. We also demonstrate the ability to nitrogen purge the flexible waveguide, increasing the power transmission by up to 20% at 2.85 THz, which paves the way for future fibre based terahertz sensing and spectroscopy applications.

Genetic dissection of Iddm26 in the spontaneously diabetic BBDP rat.

The 40 Mb T1D susceptibility locus Iddm26 was mapped to chromosome 2 through linkage analysis of a conditioned cross-intercross between the diabetes-prone BBDP and the diabetes-resistant ACI.BBDP-Iddm1,Iddm2 (ACI.1u.Lyp). It is flanked by Iddm32 and Iddm33, which control the kinetics of disease progression. To fine-map Iddm26 and characterize immune phenotypes controlled by this locus, several congenic sublines were generated carrying smaller, overlapping intervals spanning Iddm26 and fragments of Iddm32 and 33. Analysis of disease susceptibility, age of disease onset, and immune phenotypes in these sublines identified subloci regulating these different parameters. Two ACI.1u.Lyp-derived subloci, Iddm26.1 and Iddm26.2, imparted significant protection from diabetes, decreasing the cumulative incidence by as much as 57% and 28%, respectively. Iddm26.2, which overlaps with the human PTPN22 locus, only affected disease susceptibility, whereas Iddm26.1 also significantly affected disease kinetics, delaying T1D onset by more than 10 days compared with the parental BBDP strain. These Iddm26 subloci also regulated various immune phenotypes, including the proportion of splenic macrophages by Iddm26.1, and the proportion of activated T-cells in secondary lymphoid organs by Iddm26.2. The analysis of Iddm26 congenic animals in two different SPF facilities demonstrated that the influence of this locus on T1D is environment-dependent.

Perspectives from the 2nd International Post-Tuberculosis Symposium: mobilising advocacy and research for improved outcomes.

In 2020, it was estimated that there were 155 million survivors of TB alive, all at risk of possible post TB disability. The 2nd International Post-Tuberculosis Symposium (Stellenbosch, South Africa) was held to increase global awareness and empower TB-affected communities to play an active role in driving the agenda. We aimed to update knowledge on post-TB life and illness, identify research priorities, build research collaborations and highlight the need to embed lung health outcomes in clinical TB trials and programmatic TB care services. The symposium was a multidisciplinary meeting that included clinicians, researchers, TB survivors, funders and policy makers. Ten academic working groups set their own goals and covered the following thematic areas: 1) patient engagement and perspectives; 2) epidemiology and modelling; 3) pathogenesis of post-TB sequelae; 4) post-TB lung disease; 5) cardiovascular and pulmonary vascular complications; 6) neuromuscular & skeletal complications; 7) paediatric complications; 8) economic-social and psychological (ESP) consequences; 9) prevention, treatment and management; 10) advocacy, policy and stakeholder engagement. The working groups provided important updates for their respective fields, highlighted research priorities, and made progress towards the standardisation and alignment of post-TB outcomes and definitions.

En 2020, il est estimé qu'il y a 155 millions de survivants de la TB dans le monde, tous exposés à un risque d'invalidité post-TB. Le deuxième Symposium International Post-Tuberculose (Stellenbosch, Afrique du Sud) a été organisé dans le but de sensibiliser davantage à l'échelle mondiale et de permettre aux communautés touchées par la TB de contribuer activement à la mise en œuvre de l'agenda. De plus, nous avons entrepris de mettre à jour les connaissances sur la vie et les maladies post-TB, de déterminer les domaines de recherche prioritaires, d'établir des partenariats de recherche et de souligner l'importance d'intégrer les résultats sur la santé pulmonaire dans les essais cliniques et les services de soins de la TB. Le symposium était une réunion de travail pluridisciplinaire rassemblant des praticiens, des chercheurs, des personnes ayant survécu à la TB, des donateurs, des décideurs politiques et d'autres acteurs clés. Dix groupes de travail académiques ont établi leurs propres objectifs et ont abordé les sujets thématiques suivants : 1) engagement et perspectives des patients ; 2) épidémiologie et modélisation ; 3) pathogénie des séquelles post-TB ; 4) maladie pulmonaire post-TB (PTLD, pour l'anglais «post-TB lung disease ¼) ; 5) complications cardiovasculaires et vasculaires pulmonaires ; 6) complications neuromusculaires et squelettiques ; 7) complications pédiatriques ; 8) conséquences économiques, sociales et psychologiques (ESP, pour l'anglais «economic-social and psychological¼) ; 9) prévention, traitement et gestion ; 10) plaidoyer, politique et engagement des parties prenantes. Les groupes de travail académiques ont apporté des mises à jour significatives dans leurs domaines respectifs, ont mis en évidence les priorités de recherche et ont avancé vers la normalisation et l'harmonisation des résultats et des définitions de la post-TB.

Mannan binding lectin-associated serine protease 1 is induced by hepatitis C virus infection and activates human hepatic stellate cells.

Mannan binding lectin (MBL)-associated serine protease type 1 (MASP-1) has a central role in the lectin pathway of complement activation and is required for the formation of C3 convertase. The activity of MASP-1 in the peripheral blood has been identified previously as a highly significant predictor of the severity of liver fibrosis in hepatitis C virus (HCV) infection, but not in liver disease of other aetiologies. In this study we tested the hypotheses that expression of MASP-1 may promote disease progression in HCV disease by direct activation of hepatic stellate cells (HSCs) and may additionally be up-regulated by HCV. In order to do so, we utilized a model for the maintenance of primary human HSC in the quiescent state by culture on basement membrane substrate prior to stimulation. In comparison to controls, recombinant MASP-1 stimulated quiescent human HSCs to differentiate to the activated state as assessed by both morphology and up-regulation of HSC activation markers α-smooth muscle actin and tissue inhibitor of metalloproteinase 1. Further, the expression of MASP-1 was up-regulated significantly by HCV infection in hepatocyte cell lines. These observations suggest a new role for MASP-1 and provide a possible mechanistic link between high levels of MASP-1 and the severity of disease in HCV infection. Taken together with previous clinical observations, our new findings suggest that the balance of MASP-1 activity may be proinflammatory and act to accelerate fibrosis progression in HCV liver disease.

Development and validation of a real-time PCR assay for the detection of Aeromonas salmonicida.

A real-time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10(4) ± 1 × 10(4) CFU g(-1) (without enrichment) and 40 ± 10 CFU g(-1) (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real-time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real-time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.

Performance of spirometry assessment at TB diagnosis.

BACKGROUND: Spirometry is considered relevant for the diagnosis and monitoring of post-TB lung disease. However, spirometry is rarely done in newly diagnosed TB patients.METHODS: Newly diagnosed, microbiologically confirmed TB patients were recruited for the study. Spirometry was performed within 21 days of TB treatment initiation according to American Thoracic Society/European Respiratory Society guidelines. Spirometry analysis was done using Global Lung Initiative equations for standardisation.RESULTS: Of 1,430 eligible study participants, 24.7% (353/1,430) had no spirometry performed mainly due to contraindications and 23.0% (329/1,430) had invalid results; 52.3% (748/1,430) of participants had a valid result, 82.8% (619/748) of whom had abnormal spirometry. Of participants with abnormal spirometry, 70% (436/619) had low forced vital capacity (FVC), 6.1% (38/619) had a low ratio of forced expiratory volume in 1 sec (FEV1) to FVC, and 19.1% (118/619) had low FVC, as well as low FEV1/FVC ratio. Among those with abnormal spirometry, 26.3% (163/619) had severe lung impairment.CONCLUSIONS: In this population, a high proportion of not performed and invalid spirometry assessments was observed; this was addressed by removing tachycardia as a (relative) contraindication from the study guidance and retraining. The high proportion of patients with severe pulmonary impairment at the time of TB diagnosis suggests a huge morbidity burden and calls for further longitudinal studies on the relevance of spirometry in predicting chronic lung impairment after TB.

A phase IIb, open-label, randomized controlled dose ranging multi-centre trial to evaluate the safety, tolerability, pharmacokinetics and exposure-response relationship of different doses of delpazolid in combination with bedaquiline delamanid moxifloxacin in adult subjects with newly diagnosed, uncomplicated, smear-positive, drug-sensitive pulmonary tuberculosis.

BACKGROUND: Linezolid is an effective, but toxic anti-tuberculosis drug that is currently recommended for the treatment of drug-resistant tuberculosis. Improved oxazolidinones should have a better safety profile, while preserving efficacy. Delpazolid is a novel oxazolidinone developed by LegoChem Biosciences Inc. that has been evaluated up to phase 2a clinical trials. Since oxazolidinone toxicity can occur late in treatment, LegoChem Biosciences Inc. and the PanACEA Consortium designed DECODE to be an innovative dose-ranging study with long-term follow-up for determining the exposure-response and exposure-toxicity relationship of delpazolid to support dose selection for later studies. Delpazolid is administered in combination with bedaquiline, delamanid and moxifloxacin. METHODS: Seventy-five participants with drug-sensitive, pulmonary tuberculosis will receive bedaquiline, delamanid and moxifloxacin, and will be randomized to delpazolid dosages of 0 mg, 400 mg, 800 mg, 1200 mg once daily, or 800 mg twice daily, for 16 weeks. The primary efficacy endpoint will be the rate of decline of bacterial load on treatment, measured by MGIT liquid culture time to detection from weekly sputum cultures. The primary safety endpoint will be the proportion of oxazolidinone class toxicities; neuropathy, myelosuppression, or tyramine pressor response. Participants who convert to negative liquid media culture by week 8 will stop treatment after the end of their 16-week course and will be observed for relapse until week 52. Participants who do not convert to negative culture will receive continuation phase treatment with rifampicin and isoniazid to complete a six-month treatment course. DISCUSSION: DECODE is an innovative dose-finding trial, designed to support exposure-response modelling for safe and effective dose selection. The trial design allows assessment of occurrence of late toxicities as observed with linezolid, which is necessary in clinical evaluation of novel oxazolidinones. The primary efficacy endpoint is the change in bacterial load, an endpoint conventionally used in shorter dose-finding trials. Long-term follow-up after shortened treatment is possible through a safety rule excluding slow-and non-responders from potentially poorly performing dosages. TRIAL REGISTRATION: DECODE was registered in ClinicalTrials.gov before recruitment start on 22 October 2021 (NCT04550832).

Development and validation of real-time PCR for the detection of Yersinia ruckeri.

Yersiniosis (enteric red mouth disease) is a contagious bacterial disease caused by Yersinia ruckeri, which primarily affects salmonids. A real-time PCR assay using a molecular beacon has been developed and validated to improve the detection of the causative biotypes of Y. ruckeri. The assay, which targets the glnA (glutamine synthetase) gene, proved to have 100% analytical specificity and analytical sensitivities of 5 fg and 3 × 10(3) CFU g(-1) for DNA and seeded kidney tissue, respectively. The assay was highly repeatable with low % CV for intra- and inter-run experiments, and the optimized parameters transferred easily between different real-time PCR platforms. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon (n = 750) from 10 farms during 2007/08. The real-time PCR was run in parallel with the bacterial culture detection method, and all fish tested were found to be negative by both methods for Y. ruckeri, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive, reproducible and a rapid method for the detection of Y. ruckeri and has the potential to be used for routine diagnostic testing, health certification and active surveillance programmes.

Clinical standards for the assessment, management and rehabilitation of post-TB lung disease.

BACKGROUND: Increasing evidence suggests that post-TB lung disease (PTLD) causes significant morbidity and mortality. The aim of these clinical standards is to provide guidance on the assessment and management of PTLD and the implementation of pulmonary rehabilitation (PR).METHODS: A panel of global experts in the field of TB care and PR was identified; 62 participated in a Delphi process. A 5-point Likert scale was used to score the initial ideas for standards and after several rounds of revision the document was approved (with 100% agreement).RESULTS: Five clinical standards were defined: Standard 1, to assess patients at the end of TB treatment for PTLD (with adaptation for children and specific settings/situations); Standard 2, to identify patients with PTLD for PR; Standard 3, tailoring the PR programme to patient needs and the local setting; Standard 4, to evaluate the effectiveness of PR; and Standard 5, to conduct education and counselling. Standard 6 addresses public health aspects of PTLD and outcomes due to PR.CONCLUSION: This is the first consensus-based set of Clinical Standards for PTLD. Our aim is to improve patient care and quality of life by guiding clinicians, programme managers and public health officers in planning and implementing adequate measures to assess and manage PTLD.

The risk of tuberculosis related to tumour necrosis factor antagonist therapies: a TBNET consensus statement.

Anti-tumour necrosis factor (TNF) monoclonal antibodies or soluble TNF receptors have become an invaluable treatment against chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. Individuals who are treated with TNF antagonists are at an increased risk of reactivating latent infections, especially tuberculosis (TB). Following TNF antagonist therapy, the relative risk for TB is increased up to 25 times, depending on the clinical setting and the TNF antagonist used. Interferon-γ release assays or, as an alternative in individuals without a history of bacille Calmette-Guérin vaccination, tuberculin skin testing is recommended to screen all adult candidates for TNF antagonist treatment for the presence of latent infection with Mycobacterium tuberculosis. Moreover, paediatric practice suggests concomitant use of both the tuberculin skin test and an interferon-γ release assay, as there are insufficient data in children to recommend one test over the other. Consequently, targeted preventive chemotherapy is highly recommended for all individuals with persistent M. tuberculosis-specific immune responses undergoing TNF antagonist therapy as it significantly reduces the risk of progression to TB. This TBNET consensus statement summarises current knowledge and expert opinions and provides evidence-based recommendations to reduce the TB risk among candidates for TNF antagonist therapy.

Targeted deletions of complement lectin pathway genes improve outcome in traumatic brain injury, with MASP-2 playing a major role.

The lectin pathway (LP) of complement activation is believed to contribute to brain inflammation. The study aims to identify the key components of the LP contributing to TBI outcome as possible novel pharmacological targets. We compared the long-term neurological deficits and neuropathology of wild-type mice (WT) to that of mice carrying gene deletions of key LP components after experimental TBI. WT or MASP-2 (Masp2-/-), ficolin-A (Fcna-/-), CL-11 (Colec11-/-), MASP-1/3 (Masp1-/-), MBL-C (Mbl2-/-), MBL-A (Mbl1-/-) or MBL-/- (Mbl1-/-/Mbl2-/-) deficient male C57BL/6J mice were used. Mice underwent sham surgery or TBI by controlled cortical impact. The sensorimotor response was evaluated by neuroscore and beam walk tests weekly for 4 weeks. To obtain a comparative analysis of the functional outcome each transgenic line was rated according to a health score calculated on sensorimotor performance. For selected genotypes, brains were harvested 6 weeks after injury for histopathological analysis. MASP-2-/-, MBL-/- and FCN-A-/- mice had better outcome scores compared to WT. Of these, MASP-2-/- mice had the best recovery after TBI, showing reduced sensorimotor deficits (by 33% at 3 weeks and by 36% at 4 weeks). They also showed higher neuronal density in the lesioned cortex with a 31.5% increase compared to WT. Measurement of LP functional activity in plasma from MASP-2-/- mice revealed the absence of LP functional activity using a C4b deposition assay. The LP critically contributes to the post-traumatic inflammatory pathology following TBI with the highest degree of protection achieved through the absence of the LP key enzyme MASP-2, underlining a therapeutic utility of MASP-2 targeting in TBI.

Post-tuberculosis lung health: perspectives from the First International Symposium.

ALTHOUGH CURABLE, TB frequently leaves the individual with chronic physical and psycho-social impairment, but these consequences have been largely neglected. The 1st International Post-Tuberculosis Symposium (Stellenbosch, South Africa) was held to discuss priorities and gaps in addressing this issue. A barrier to progress has been the varied terminology and nomenclature, so the Delphi process was used to achieve consensus on definitions. Lack of sufficient evidence hampered definitive recommendations in most domains, including prevention and treatment of post-TB lung disease (PTLD), but the discussions clarified the research needed. A consensus was reached on a toolkit for future PTLD measurement and on PTLD patterns to be considered. The importance of extra-pulmonary consequences and progressive impairment throughout the life-course was identified, including TB recurrence and increased mortality. Patient advocates emphasised the need to address the psychological and social impacts post TB and called for clinical guidance. More generally, there is an urgent need for increased awareness and research into post-TB complications.

Mutations that improve efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome.

New enzymes often evolve by gene amplification and divergence. Previous experimental studies have followed the evolutionary trajectory of an amplified gene, but have not considered mutations elsewhere in the genome when fitness is limited by an evolving gene. We have evolved a strain of Escherichia coli in which a secondary promiscuous activity has been recruited to serve an essential function. The gene encoding the 'weak-link' enzyme amplified in all eight populations, but mutations improving the newly needed activity occurred in only one. Most adaptive mutations occurred elsewhere in the genome. Some mutations increase expression of the enzyme upstream of the weak-link enzyme, pushing material through the dysfunctional metabolic pathway. Others enhance production of a co-substrate for a downstream enzyme, thereby pulling material through the pathway. Most of these latter mutations are detrimental in wild-type E. coli, and thus would require reversion or compensation once a sufficient new activity has evolved.

Identification of antigens of Mycobacterium tuberculosis using human monoclonal antibodies.

Pleural tuberculosis constitutes a human model of local protective immunity to mycobacterial infection as the disease is usually self-limited and recurrent pleurisy is rare. To identify potentially protective antigens of Mycobacterium tuberculosis, 37 human pleural fluid B cell clones were established using EBV and their supernatants assayed by ELISA and Western blot for antibody reactivity with M. tuberculosis sonicate and culture filtrate. One antibody identified 29,000, 31,000, and 33,000 bands in culture filtrate, and 31,000, 33,000, and 47,000 bands in sonicate; its species reactivity by ELISA was limited to M. tuberculosis. Eight antibodies identified a 31,000 band in culture filtrate and a 68,000 band in M. tuberculosis sonicate, suggesting recognition of a secreted antigen. The species crossreactivity of these eight antibodies extended to M. avium. Six antibodies identified multiple bands and had crossreactivity that included M. avium and M. kansasii. There was no reactivity with recombinant M. tuberculosis 65,000 antigen. Tuberculous pleurisy may prove useful in the identification of potentially protective mycobacterial antigens, particularly those secreted during active infection, and thus accessible to the human immune response.

Cell membrane is a major locus for ultraviolet B-induced alterations in accessory cells.

In vitro ultraviolet B (UVB) irradiation of human blood monocytes inhibits their accessory cell function for antigen- and mitogen-induced T cell responses. These studies were designed to characterize the nature of the UVB-induced defect in human monocyte accessory cell function. Irradiated monocytes were deficient in their ability to serve as accessory cells for OKT3-induced T cell activation. In vitro exposure of monocytes to 100 J/m2 UVB completely inhibited the T cell proliferative response (51502 cpm, non-UVB-irradiated; 302 cpm, UVB-irradiated). Analysis of the accessory signals altered by UVB indicated that irradiated monocytes were incapable of binding to OKT3 molecules attached to the CD3 antigen on T cells. Provision of an alternative mechanism for binding of OKT3 molecules by attaching anti-mouse IgG to the bottom of microtiter wells completely restored accessory cell function. Further characterization of the defect demonstrated that UVB radiation did not deplete p72 Fc receptors from the surface of irradiated monocytes. However, UVB exposure did produce a dose-dependent decrease in monocyte membrane expression of ICAM-1. It is proposed that UVB radiation leads to changes within the cell membrane that inhibit the ability of monocytes to express selected molecules necessary for binding of T cells.

Induction of monocyte expression of tumor necrosis factor alpha by the 30-kD alpha antigen of Mycobacterium tuberculosis and synergism with fibronectin.

Native 30-kD antigen, also known as alpha antigen, is a fibronectin-binding protein that is secreted by live Mycobacterium tuberculosis. This antigen may play an important biological role in the host-parasite interaction since it elicits delayed type hypersensitivity response and protective immunity in vivo and T lymphocyte blastogenesis and IFN-gamma production in vitro. In the present study, we show that, TNF-alpha protein is produced in monocyte culture supernatants in response to 30-kD antigen and the level is as high as that to purified protein derivative of M. tuberculosis. This stimulatory effect was not due to contamination with either bacterial lipopolysaccharide or mycobacterial lipoarabinomannan. The preincubation of monocytes with plasma fibronectin significantly enhanced the release of TNF-alpha into the culture supernatants in response to 30-kD antigen. This effect was blocked by polygonal antibody to plasma fibronectin. In contrast, the monocytic cell line U937 failed to release TNF-alpha protein in the culture supernatants in response to 30-kD antigen with or without preincubation with plasma fibronectin. To determine whether this observation was due to differential binding of the 30-kD to fibronectin on these cells, a cell based ELISA was used. Pretreatment of monocytes with fibronectin enhanced their binding of the 30-kD antigen. U937 cells bound the 30-kD antigen weakly with or without fibronectin pretreatment. These results indicate that 30-kD antigen which is a known secretary antigen of M. tuberculosis is a stimulus for human monocytes to express TNF-alpha and that stimulatory effect may be mediated through plasma fibronectin.