Passive intranasal monoclonal antibody prophylaxis against murine Pneumocystis carinii pneumonia

Passive antibody immunoprophylaxis is one method used to protect patients against infection if they are unable to mount an adequate active immune response. Topical application of antibody may be effective against infections at mucosal sites. Using a SCID mouse model of Pneumocystis carinii pneumonia, we were able to demonstrate protection against an airborne challenge with P. carinii by intranasal administration of antibody. Immunoglobulin M (IgM) monoclonal antibodies to an epitope shared by mouse and human P. carinii organisms reduced organism numbers by more than 99% under the conditions described. An IgG1 switch variant of one of the IgM monoclonal antibodies was also protective. These experiments provide a model for exploring the utility of this approach in protecting at-risk patients from infection with P. carinii.     

Neutrophil Migration in Opposing Chemoattractant Gradients Using Microfluidic Chemotaxis Devices

Neutrophils migrating in tissue respond to complex overlapping signals generated by a variety of chemotactic factors (CFs). Previous studies suggested a hierarchy between bacteria-derived CFs and host-derived CFs but could not differentiate neutrophil response to potentially equal host-derived CFs (IL-8 and LTB₄). This paper reports neutrophil migration in conflicting gradients of IL-8 and LTB₄ using a microfluidic chemotaxis device that can generate stable and well-defined gradients. We quantitatively characterized the movement of cells from time-lapse images. Neutrophils migrate more efficiently toward single IL-8 gradients than single LTB₄ gradients as measured by the effective chemotactic index (ECI). In opposing gradients of IL-8 and LTB₄, neutrophils show obvious chemotaxis toward a distant gradient, consistent with previous reports. When an opposing gradient of LTB₄ is present, neutrophils show less effective chemotaxis toward IL-8 than when they are in a gradient of IL-8 alone. In contrast, the chemotactic response of neutrophils to LTB₄ is not reduced in opposing gradients as compared to that in a single LTB₄ gradient. These results indicate that the presence of one host-derived CF modifies the response of neutrophils to a second CF suggesting a subtle hierarchy between them.This revised version was published online in May 2005 with corrected author affiliations     

High rates of clustering of strains causing tuberculosis in Harare, Zimbabwe: a molecular epidemiological study

We examined the pattern of tuberculosis (TB) transmission (i.e., reactivation versus recent transmission) and the impact of human immunodeficiency virus (HIV) infection in Harare, Zimbabwe. Consecutive adult smear-positive pulmonary TB patients presenting to an urban hospital in Harare were enrolled. A detailed epidemiological questionnaire was completed, and tests for HIV type 1 and CD4 cell counts were performed for each patient. Molecular fingerprinting of the genomic DNA recovered from cultures of sputum was performed by two molecular typing methods: spacer oligonucleotide typing (spoligotyping) and analysis of variable number of tandem DNA repeats (VNTRs). A cluster was defined as isolates from two or more patients that shared the same spoligotype pattern or the same VNTR pattern, or both. DNA suitable for typing was recovered from 224 patients. The prevalence of HIV infection was 79%. Of 187 patient isolates (78.6%) typed by both spoligotyping and analysis of VNTRs, 147 were identified as part of a cluster by both methods. By spoligotyping alone, 84.1% of patient isolates were grouped into 20 clusters. The cluster size was generally <8 patient isolates, although three large clusters comprised 68, 25, and 23 patient isolates. A total of 89.4% of the patient isolates grouped into 12 clusters defined by analysis of VNTRs, with 2 large clusters consisting of 127 and 13 patient isolates, respectively. Thirty-six percent of patient isolates with a shared spoligotype and 17% with a shared VNTR pattern were geographically linked within Harare, but they were not linked on the basis of the patient's home district. In a multivariate analysis, there were no independent predictors of clustering, including HIV infection status. Comparison with the International Spoligotype database (Pasteur Institute, Pointe a Pitre, Guadeloupe) demonstrated that our three largest spoligotype clusters are well recognized and ubiquitous in Africa. In this epidemiologically well characterized urban population with a high prevalence of HIV infection, we identified a very high level of strain clustering, indicating substantial ongoing recent TB transmission. Geographic linkage could be detected in a proportion of these clusters. A small group of actively circulating strains accounted for most of the cases of TB transmission.     

Generation of CD8(+) T-cell responses to Mycobacterium bovis and mycobacterial antigen in experimental bovine tuberculosis

Protective immunity against tuberculosis is considered to be essentially cell mediated, and an important role for CD8(+) T lymphocytes has been suggested by several studies of murine and human infections. The present work, using an experimental model of infection with Mycobacterium bovis in cattle, showed that live M. bovis elicits the activation of CD8(+) T cells in vitro. However, a sonic extract prepared from M. bovis (MBSE) and protein purified derivative (PPDb) also induced a considerable degree of activation of the CD8(+) T cells. Analysis of proliferative responses of peripheral blood mononuclear cells, purified CD8(+) T cells, and CD8(+) T-cell clones to M. bovis and to soluble antigenic preparations (MBSE, PPDb) showed that the responses of all three types of cells were always superior for live mycobacteria but that strong responses were also obtained with complex soluble preparations. Furthermore, while cytotoxic capabilities were not investigated, the CD8(+) T cells were found to produce and release gamma interferon in response to antigen (live and soluble), which indicated one possible protective mechanism for these cells in bovine tuberculosis. Finally, it was demonstrated by metabolic inhibition with brefeldin A and cytochalasin D at the clonal level that an endogenous pathway of antigen processing is required for presentation to bovine CD8(+) cells and that presentation is also dependent on phagocytosis of the antigen.     

Immunotherapy of mammary adenocarcinoma metastases in C3H/HeN mice with chronic administration of cyclo-oxygenase inhibitors alone or in combination with IL-2

In this study the efficacy of treatment of two cyclo-oxygenase inhibitors, ibuprofen (Ibu) and indomethacin (Indo), are compared in the immunotherapy of metastasis designed to reverse prostaglandin E₂ (PGE₂)mediated inactivation of interleukin-2 (IL-2)-dependent host killer cell lineages. These agents were tested either alone for the prevention of metastasis or in combination with IL-2 for the eradication of established metastasis. C3H/HeN mice were placed on chronic oral Ibu (CIbT; 200 and 600 ,g/ml of water) or Indo (CIT; 10 g/ml) 5 days after s.c. transplantation of 5 × 10⁵ metastatic C3L5 mammary carcinoma for the prevention of spontaneous lung metastases. They showed intolerance to Indo at a dosage of 14 g/ml, which was well tolerated by other mouse strains in previous studies, but tolerated the Ibu dosages used. Control and treated mice were killed on day 30 to score metastatic lung colonies, to evaluate killer activity in splenocytes against natural killer (NK)-sensitive YAC-1 lymphoma or NK-resistant C3L5 adenocarcinoma and 8911 lymphoma targets, and to phenotype the surface markers of killer cells. CIbT and CIT alone at the above dosage significantly reduced the number of lung colonies, retarded local tumor growth and restored NK activity of splenic killer cells expressing AGM-1⁺, Thy-1^–, Lyt-2^– phenotype. To treat established lung metastasis, mice bearing 15-day C3L5 transplants were given CIbT or CIT alone or in combination with two 4-day rounds (days 20–23, 31–34) of IL-2 (15 000 Cetus units, i.p. every 8 h) and were killed on day 35 to score lung colonies and characterize splenic killer cells. CIbT or CIT alone reduced the number of spontaneous lung metastases and restored anti-YAC-1 killer function of splenocytes with NK-like phenotype (AGM-1⁺, Thy-1^–, Lyt-2^–); some anti-C3L5 killer function was also generated in the high dose Ibu group and the killer cell showed AGM-1⁺, Thy-1⁺ and Lyt-2⁺ phenotype. Combined therapies with CIbT or CIT plus IL-2 were more effective in reducing metastases and promoting killer cell function, the best results being achieved with high dose Ibu + IL-2. All killer cells expressed AGM-1 and Thy-1. In addition, C3L5 killer cells also expressed Lyt-2, suggesting T-cell stimulation. PGE₂ synthesis in the host was inhibited by at least 50% in mice subjected to CIbT or CIT. Thus, Ibu proved to be an excellent substitute for Indo in preventing metastasis and NK cell activation when given alone, and also in ameliorating established metastasis and activating lymphokine-activated killer cells when combined with IL-2.     

Employing a systematic approach to biobanking and analyzing clinical and genetic data for advancing COVID-19 research

Within the GEN-COVID Multicenter Study, biospecimens from more than 1000 SARS-CoV-2 positive individuals have thus far been collected in the GEN-COVID Biobank (GCB). Sample types include whole blood, plasma, serum, leukocytes, and DNA. The GCB links samples to detailed clinical data available in the GEN-COVID Patient Registry (GCPR). It includes hospitalized patients (74.25%), broken down into intubated, treated by CPAP-biPAP, treated with O₂ supplementation, and without respiratory support (9.5%, 18.4%, 31.55% and 14.8, respectively); and non-hospitalized subjects (25.75%), either pauci- or asymptomatic. More than 150 clinical patient-level data fields have been collected and binarized for further statistics according to the organs/systems primarily affected by COVID-19: heart, liver, pancreas, kidney, chemosensors, innate or adaptive immunity, and clotting system. Hierarchical clustering analysis identified five main clinical categories: (1) severe multisystemic failure with either thromboembolic or pancreatic variant; (2) cytokine storm type, either severe with liver involvement or moderate; (3) moderate heart type, either with or without liver damage; (4) moderate multisystemic involvement, either with or without liver damage; (5) mild, either with or without hyposmia. GCB and GCPR are further linked to the GCGDR, which includes data from whole-exome sequencing and high-density SNP genotyping. The data are available for sharing through the Network for Italian Genomes, found within the COVID-19 dedicated section. The study objective is to systematize this comprehensive data collection and begin identifying multi-organ involvement in COVID-19, defining genetic parameters for infection susceptibility within the population, and mapping genetically COVID-19 severity and clinical complexity among patients.Subject terms: Genetics research, Viral infection     

Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon

Animal studies with Streptococcus pneumoniae have provided valuable models for drug development. In order to monitor long-term pneumococcal infections noninvasively in living mice, a novel gram-positive lux transposon cassette, Tn4001 luxABCDE Km(r), that allows random integration of lux genes onto the bacterial chromosome was constructed. The cassette was designed so that the luxABCDE and kanamycin resistance genes were linked to form a single promoterless operon. Bioluminescence and kanamycin resistance only occur in a bacterial cell if this operon has transposed downstream of a promoter on the bacterium's chromosome. S. pneumoniae D39 was transformed with plasmid pAUL-A Tn4001 luxABCDE Km(r), and a number of highly bioluminescent colonies were recovered. Genomic DNA from the brightest D39 strain was used to transform a number of clinical S. pneumoniae isolates, and several of these strains were tested in animal models, including a pneumococcal lung infection model. Strong bioluminescent signals were seen in the lungs of the animals containing these pneumococci, allowing the course and antibiotic treatment of the infections to be readily monitored in real time in the living animals. Recovery of the bacteria from the animals showed that the bioluminescent signal corresponded to the number of CFU and that the lux construct was highly stable even after several days in vivo. We believe that this lux transposon will greatly expand the ability to evaluate drug efficacy against gram-positive bacteria in living animals using bioluminescence.     

Use of polymerase chain reaction for rapid diagnosis of tuberculosis.

A DNA amplification assay using the polymerase chain reaction technique designed for the rapid identification of Mycobacterium bovis organisms was used to test 211 human mycobacterial isolates and 177 clinical specimens previously submitted for routine mycobacterial culture. The procedures described could be used by routine or specialist laboratories for identification of M. tuberculosis complex organisms in 4 h and/or as a rapid screening method for the direct detection of M. tuberculosis complex organisms in specimens.     

Knowledge,attitudes and practices towards HIV positive and AIDS patients among public service dentists in Nairobi

The purpose of this study was to assess the knowledge, practices and attitudes towards HIV Positive/AIDS patients among 112 dentists from public institutions in Nairobi using a self-administered questionnaire. 94(83.9%) responded. Over 74% had managed HIV positive/AIDS patients. In general, respondents' knowledge and preventive measures against HIV infection were satisfactory. 8.5% did not find use of protective eye wear absolutely necessary. 33% used protective covers routinely. 52.1% advocated for the screening of all suspected cases of AIDS before treatment. 27.7% felt that HIV positive health workers and those with AIDS should not be allowed to treat patients. 53.2% felt that they should be given the right to decide on treating HIV Positive/AIDS patients. 10.6% supported the idea that AIDS patients be isolated from uninfected individuals. It is concluded that a substantial number of dentists were wanting in their attitudes towards HIV positive/AIDS patients.     

The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in different species

Ataxia-telangiectasia (A-T) is an autosomal recessive disorderinvolving cerebellar degeneration, immunodeficiency, radiationsensitivity, and cancer predisposition. A-T heterozygotes aremoderately cancer prone. The A-T gene, designated ATM, was recentlyidentified in our laboratory by positional cloning, and a partialcDNA clone was found to encode a polypeptide with a PI-3 kinasedomain. We report here the molecular cloning of a cDNA contigspanning the complete open reading frame of the ATM gene. Thepredicted protein of 3056 amino acids shows significant sequencesimilarities to several large proteins in yeast, Drosophilaand mammals, all of which share the PI-3 kinase domain. Manyof these proteins are involved in the detection of DNA damageand the control of cell cycle progression. Mutations in theirgenes confer a variety of phenotypes with features similar tothose observed in human A-T cells. The complete sequence ofthe ATM gene product provides useful clues to the function ofthis protein, and furthers understanding of the pleiotropicnature of the A-T mutations.