Streptococcus Pneumoniae Treatment Testing
A new drug that improves macrophage performance was to be tested for the treatment of pneumonia in humans by the bacterium Streptococcus pneumoniae. As an animal model would be required for phase 1 clinical trial, suitability of animal and test bacterium and the test procedures were to be envisaged carefully to emulate the outcome of the experiments for human application. A careful persuasion of the project would take one year and with a principal investigator and two assistants – an animal housekeeper and scientist. The budget would be for obtaining animal and bacterial strains, for approval from the ethical committee (contingency), for rearing the animals (infrastructure), for analysis of all parameters related to pneumonia (consumables, equipment), and for final project report and publications (printing cost). Pneumonia pathogenesis has been tested in many animal models, including Sprague-Dawley rats, sheep, dogs, etc. Recently, Mizgerd and Skerrett have elaborately reviewed the rationale of using animal models for testing human pneumonia.
The mice test system has several advantages such as small size, high reproductive rates, availability of genetically engineered stem cells, knock-out mutants, and several murine proteins and antibodies, and genetically identical cohorts of inbred animals. There are also differences between humans. The murine respiratory tract is different from humans, which may influence the deposition or clearance of aspirated test bacterium. The secretary defenses owing to the epithelial lining and local phagocytic defenses, e.g., by inducible nitrite oxidase synthase, are also different. Toll-like receptors, which recognize a pathogen and trigger an innate and adaptive immune response, are different, e.g., TLR3 is highly expressed by macrophages in mice, and TLR5 is differentially expressed. There are also differences in inflammatory and immune responses, e.g., no mice counterpart of IL-8, a chemokine responsible for neutrophil adherence to the site of infection, is present.
These problems can be circumvented to a large extent by choosing the right strain of inbreeding mice. For the aforementioned infection, BALB/c exhibit no bacteremia or infection, C57BL/6 or DBA/2 show moderate and CBA/Ca, C3H/He, and SJL exhibit severe bacteremia and 100% infection. Just as mouse strain is critical, the S. pneumoniae is also, for e.g., serotype 2 and 3 cause fatality, but serotype 19 has a non-significant response. The efficacy of any drug that stimulates the macrophages has to be antigenically similar to the bacterial surface. The reason is that macrophages recognize surface peptidoglycan, lipopolysaccharides, etc., and can not differentiate inactive adjuvant cells and life pathogens. The following research program can be envisaged:
- Certification of training skills to work on animals by the “Animals (Scientific Procedures) Act” and other institutional and governmental ethical committees.
- Collection of mice (CBA/Ca, C3H/He) and inbreeding using animal house facility.
- Procurement of S. pneumoniae serotype 2 or 3 and storage at -70oC in skim milk or Todd-Hewitt broth supplemented with 0.5% yeast extract and 20% glycerol establishing growth up to 107 CFU ml-1.
- Setting up the infection by aerosol dissemination, direct endotracheal, endobronchial or intranasal instillations, transtracheal injections, or peroral incubations. With efficient methods, a large number of cells can be infected in the nasal cavity and lungs, similar to humans.
- Measuring microbial burden: Quantitative culturing is a standard method to enumerate bacterial infestations. For this, lungs are excised from euthanized mice, homogenized, and serially diluted before CFU is enumerated on the appropriate medium. Currently, an improvised method is to apply luciferase (bioluminescence) reporter gene-expressive S. pneumoniae. The illuminative bacteria from the thorax give quantitative measurements of bacterial colonization, and for this, mice need not be sacrificed.
- Measuring inflammatory response by neutrophil recruitment and pulmonary edema. The most popular method for enumeration of neutrophils is bronchoalveolar lavage. The liquid content in the lungs can be measured by the ratio of wet weight to dry weight. Edema accumulation in alveolar space can be microscopically determined and quantified by morphometry. A more sensitive method would be to inject a tracer and to examine its accumulation in the lung (ELISA for anti-albumin antibody and spectrophotometry for Evans blue dye as tracers).
- Measuring lung mechanistic by recording a decrease in tidal volume, breathing rates, etc. These indices indirectly determine the sequelae of inflammation.
- The most ethically agreed method for testing the efficacy of any drug is Dixon’s up and down method. In duplicate/triplicate, mice of the same age can be quarantined for three days before they are infected with a previously standardized dose of S. pneumoniae. For controls, we shall use a dose of the drug. Another two controls would be desirous, one in which adjuvant anti-capsular antibody is administered to activate macrophage but without any infection, and two in which saline is administered. Once we have ruled out any manifestation of the above quantitative parameters related to pneumonia in controls and significant difference (one-way ANOVA) in infected trials, the infections will be again set up, and at different intervals, a single limited dose of the drug will be administered in above numbers. Again the quantitative parameters will be recorded to evaluate significant differences from infected controls (one-way ANOVA). If there is no significant difference, then we shall gradually increase the dose until a significant difference is recorded. Conversely, if the difference already is very significant, we shall reduce to dose until we reach a level of non-significant difference. We shall require a minimum of 12 mice for setting infection trial and 12-15 mice for determining the effective concentration of the drug.
- As we expect macrophage activation due to the drug, we might record the change in certain special cells for whose surface antigens, monoclonal antibodies can be raised and used for quantification. For infected mice and controls, the relative levels of tumor necrosis factor α, Ly-6G- on granulocytes, CD4+ and CD8+ on T cells, etc., can be recorded by immunoblotting using specific primary and secondary antibodies. This way, variation in the immune response of the drug itself or drug in conjunction with bacterial cells can be worked out, and such data would b required for immunological drugs.