**5. Treatment strategies for CF**

Considering the relative abundance of CF cases (~70,000 world-wide), it is important that efforts are put forward into treating the symptoms that arise from the disease [60]. These symptoms can be very detrimental, especially in the case of serious bacterial infections and ensuing exacerbations. Initially, the treatments that were available were only focused on chest physical therapy for the buildup of airway mucus, but those efforts have evolved as current research has illuminated more on the pathophysiology of CF lung disease. An example of advanced treatment with continuing research was the initial attempts to provide gene therapy for CF. With the idea that complementing the mutated CFTR with wild-type CFTR would allow for functional ion transport, efforts were made to transfect cells with a functional copy of the gene using either a liposomal or viral vector. However, this eventually ended in failure, as the complementation strategy was unsuccessful and in some cases harmful. Specifically, adenovirus complementation caused a hyper-immune response, and DNA liposome trans‐ fection could not properly deliver the DNA to the nucleus [61]. Recent attempts are examining whether transfecting cells with mRNA coding for functional CFTR could be beneficial. However, this still requires much more research. With that in mind, treatment falls primarily into two main camps; the removal of the dessicated mucus, and the treatment of bacterial infections.

### **5.1. Treatment of mucus buildup**

seen. This all suggested that biofilm bacteria might be able to respond better to antibiotics and environmental stresses than planktonic cells, as the biofilm is not only more inherently resistant, but the bacteria interior to the film experience a lower concentration of the stressor, allowing for genetic regulations and responses [38]. Following this paper, more attempts to analyze biofilm-related genes ensued. These studies involved mostly environmental biofilms, and not those associated with human infection. Eventually, as the prevalence of biofilms in disease became better known, it became important to discover if these changes in biofilm gene

In this regard, several attempts to examine human-associated biofilm infections occurred, but it had to be verified that the gene expression difference was reliable, and not an artifact of the harvesting procedure. Some studies did well with this issue, collecting *PA* from patients and analyzing both that sample and in vitro grown cells. These differences did seem quite signif‐ icant, showing changes between the two samples that suggested a decrease in the regulatory machinery for biofilm cells rather than an upregulation of many of the biofilm associated genes (such as *muc, alg, rhl, hcn,* and *plc*) [58]. This was novel, as the biofilm conversion seemed much

Other genes that were found to be changed in the CF lung associated samples seemed to actually be associated with the acquisition of nutrients in the surrounding area. For example, one such study found that there was an increase in genes associated with arginine metabolism and the glyoxylate shunt (a process found often with the β-oxidation of lipids to acetyl-CoA) [59]. This seems to indicate that the CF lung has a higher available amount of arginine and free lipids for degradation, potentially linked to the death of epithelial cells in the lung. This same study also found the cells generally change to a sessile state, where many of the genes associated with motility and chemotaxis are downregulated. This indicates that the cells initially colonizing the CF lung are able to prosper enough that the organisms can rapidly adapt to such conditions. This same down-regulation of motility genes is often seen with the conversion to a biofilm mode of growth. Once the bacteria have shifted to the mucoid state and are able to survive the thick, dessicated mucus, then they will be able to thrive in the lung.

This can help explain why *PA* is often such a major problem for the CF patients.

Considering the relative abundance of CF cases (~70,000 world-wide), it is important that efforts are put forward into treating the symptoms that arise from the disease [60]. These symptoms can be very detrimental, especially in the case of serious bacterial infections and ensuing exacerbations. Initially, the treatments that were available were only focused on chest physical therapy for the buildup of airway mucus, but those efforts have evolved as current research has illuminated more on the pathophysiology of CF lung disease. An example of advanced treatment with continuing research was the initial attempts to provide gene therapy for CF. With the idea that complementing the mutated CFTR with wild-type CFTR would allow for functional ion transport, efforts were made to transfect cells with a functional copy

regulation were able to be transitioned over to this situation.

188 Cystic Fibrosis in the Light of New Research

more complicated than simply via a dysregulatory pathway.

**5. Treatment strategies for CF**

Even without the involvement of pathogens in the mucus plugs that develop in the CF lung, the plugs themselves can cause serious effects on the patients, specifically in their quality of life. As mentioned previously, these plugs develop from a lack of clearance of mucus associated with the pericilliary layer, resulting in a continually developing blockage and a decreased airflow that reaches the respiratory zone of the lungs. With reduced oxygen levels, the patients begin to suffer and can eventually become cyanotic. However, this is one of the more apparent symptoms of CF, and has been a focus since the disease was first identified. Here, the primary mode of treatment has been physical percussion of the patient. This is termed as chest physical therapy (CPT) and has progressed much since it was initially developed. This treatment initially involved pounding on the patient's back in such a manner as to dislodge mucus that was clinging to the bronchi [62]. After dislodging the mucus, the patient would then cough up any that was loosened, but this process in general was quite painful and not desired by patients, even when it was successful. In an effort to make this a more effective procedure, technology began to be introduced into the process. Initially, a device similar to a back-pack, was created that performed the percussion automatically, rather than rely on the doctor or medical professional. Although this is effective in providing the same amount of force each time, the percussion itself was not eliminated from the process.

Following this, different methods were employed to physically break up the mucus. These have become more advanced in recent decades. Most are reliant on sound waves or vibrations to loosen the mucus that allows for its eventual clearance. These methods include masks and vests that patients don that create vibrations and devices that convert the exhaled air of a patient into vibrations [62]. In all of these, the pressure and pain of physical percussion is eliminated, but the positive effects remain. Even some chemical treatments are prescribed to help loosen the mucus, such as aerosolized sodium bicarbonate that can help return the mucus layer to its normal pH and allow for better clearance. Physicians also routinely prescribe bronchodilators and anti-inflammatory medications to patients to both increase the functional airway passage and to help clear some of the mucus from the airways. In some extreme cases, where this use of medication is ineffective or does not keep up with the overall progression of the disease, the patients may have to endure a lung transplant [62]. While this is occasionally effective, it usually results in the eventual buildup of mucus in the lungs once again, resulting in further treatment or potentially even another transplant.

### **5.2. Treatment of bacterial infections**

While the buildup of mucus within the lungs is quite problematic, it is often not what leads to the overall mortality or morbidity of CF patients. Rather, this is due to secondary infections that arise from this buildup. As stated earlier, this mucus is a rich, largely immobile niche of complex nutrients that enmesh the bacteria. In healthy lungs, the cilia are functioning properly with a thin PCL and clearing most pathogens out of the lungs. However, with the thick mucus, this is not the case, as the cilia do not have enough physical force to push it upwards. As a result, bacteria begin to infect the area. However, the precise location in the lungs where the infection resides is important for the choice of treatment methods. In general, the lungs are divided into two zones. The first is the conductive zone, which includes many of the prelimi‐ nary branches of the bronchi and does not directly include the alveoli or any of the accompa‐ nying areas. The second is the respiratory zone, which includes many of the later branches of the airways and the alveoli. These differences are important due to the necessary changes for treatments. In the conductive zone, the tissue does not have quite as high a vascular exposure, but is much closer to the mouth. As such, infections in this area are often treated with aero‐ solized medicines. In contrast, the respiratory zone is much deeper into the lungs, but has much higher vascular exposure due to the presence of the alveoli in this zone. Treatments for this zone usually include oral or intravenous medications. While the mode of treatment is known, the actual ability to determine where the infection is occurring is dependent on a series of initial cultures from the patient, and the species present can change once treatment begins.

Once the type of treatment has been determined, the next step is determining what the effective antibiotic(s) is for the particular bacteria being targeted. This is dependent on the class of antibiotic and has to be administered in a patient-specific manner. In general, there are some that work more efficiently in certain patients than in others. For example, tobramycin, a potent aminoglycoside, has been approved by the FDA for clinical treatment of *PA* in CF patients. However, various reports have shown an impenetrability of tobramycin in biofilms, an effect that may be due to the lowered levels of oxygen within the biofilms. The ability of an antibiotic to be active on biofilm bacteria is of the utmost importance when selecting a treatment strategy [33]. As mentioned previously, biofilms generally develop more resistance to certain antibi‐ otics due to their differential metabolism, as well as penetration issues. Those at the surface of the biofilm are generally more metabolically active, while those in the middle of the biofilm projections and those at the base are generally far less active or even inactive. Ciofu et al. [63] showed that certain antibiotics will be more or less effective in these areas depending on their mode of action. Tobramycin is highly effective on the more oxygen-rich outer layer of metabolically active bacteria, but others such as colistin are better suited at dealing with the metabolically inactive bacteria further into the biofilm. In addition, both basic and clinical scientists are focusing better on the proper physiology of bacteria for treatment. This is something that is case-dependent, and cannot necessarily be generalized.

However, the efficiency of the antibiotics for long-term treatment of *PA* infections has been worrisome, especially because of the development of multi-drug resistant *PA* (MRPA) [64]. Because of these issues, it is imperative to unravel new treatments that will work on bacteria that are growing under non-traditional conditions (e.g., anaerobic or microaerobic). That is why certain groups are evaluating the ability of other compounds for bactericidal action. One of the more notable treatments was discovered by our group in defining the use of acidified nitrite for the treatment of mucoid *PA* infections. In 2006, we discovered that the application of acidified nitrite onto anaerobic *PA*, both planktonic and biofilm associated, virtually killed the organisms; however, the same effect was not quite seen aerobically [65]. The treatment also had to be administered at a slightly acidic pH (6.5) of the CF lung. This combination, however, was very effective. Further investigation showed that acidified nitrite was unstable and produced a toxic, bactericidal intermediate nitric oxide (NO). Although *PA* normally proceeds through the route of denitrification, this process is overloaded by an excess of NO2 - , and subsequently NO. The enzymes involved in the detoxification process are not able to com‐ pensate for this overload, and the bacteria ultimately become poisoned. This occurs through the reaction of NO with sulfur from cysteines in the proteins in the cell, which produces Snitrosylated proteins (S-NO) that are targeted for degradation. In addition, NO also reacts with iron/heme containing proteins leading to the formation of dinitrosyl iron complexes. Consid‐ ering the simplicity and remarkable efficacy of this treatment, this is an example of what to look for in the future.
