Background Coccidioidomycosis or Valley Fever is caused by  Coccidioides  in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in  Coccidioides  infection are not known.
Background
Coccidioidomycosis or Valley Fever is caused by  Coccidioides  in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in  Coccidioides  infection are not known.CoccidioidesCoccidioidesMethods In this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of  Coccidioides posadasii  infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of  C. posadasii  (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays.
Methods
In this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of  Coccidioides posadasii  infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of  C. posadasii  (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays.Coccidioides posadasiiC. posadasiiResults We found that in lavage fluid samples of  C. posadasii  infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % (SEM 3.5, p < 0.001), 38 % (SEM 5.8, p < 0.001) and 4 % (SEM 1.3, p < 0.001) of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of  C. posadasii  protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens.
Results
We found that in lavage fluid samples of  C. posadasii  infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % (SEM 3.5, p < 0.001), 38 % (SEM 5.8, p < 0.001) and 4 % (SEM 1.3, p < 0.001) of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of  C. posadasii  protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens.C. posadasiiC. posadasiiConclusion Our results suggest that the  C. posadasii  infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.
Conclusion
Our results suggest that the  C. posadasii  infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.C. posadasii

Background Coccidioidomycosis or Valley Fever is a fungal disease caused by the biphasic, highly virulent, soil-fungus  Coccidioides immitis  or  posadasii  [ 1 ]. It is endemic in the southwest regions of US, Northern Mexico and parts of Central America [ 2 ].  C. posadasii or C. immitis , are the most virulent fungal pathogens enlisted in Select Agent list and pose a risk for bioterrorism [ 3 ]. The primary infection is acquired by inhalation of air-borne, mycelial phase arthroconidium that converts into endosporulating spherule in the lung. Clinical manifestations of the disease range from pulmonary infection to a more severe fatal mycosis involving extra-pulmonary tissues in 1–10% of the infected people [ 1 - 4 ]. Previous studies suggest that Th1 cell mediated immunity protects individuals against  Coccidioides  [ 5 , 6 ]. However, information is lacking regarding the pulmonary innate immune components that may play a critical role in regulation of immune responses against  Coccidioides . At alveolar level in the lung, the innate immune system is composed of many cell types and chemical mediators, including surfactant. The pulmonary surfactant is a complex mixture of lipids (88–90%) and proteins (10–12%), synthesized by type II epithelial cells and Clara cells. It lines the alveoli, and helps in maintaining normal lung function [ 7 ]. Among four different surfactant proteins, surfactant proteins-A (SP-A) and D (SP-D) are members of the "Collectin" family [ 8 ]. In the past, several studies have suggested that both SP-A and SP-D play an important role in innate host defense against various viral, fungal and bacterial pathogens [ 9 , 10 ]. More evidence for the pulmonary collectins' role in host defense comes from studies on SP-A- deficient mice that are susceptible to intra-tracheal Group B  Streptococci  [ 11 ],  Pseudomonas aeruginosa  [ 12 ], and Respiratory Syncytial Virus [ 13 ]. Also, intranasally administered SP-D has been found to reduce replication of Respiratory Syncytial Virus in the lungs of infected mice [ 14 ]. Both SP-A and SP-D, have been classified as secretory pattern-recognition receptors that can bind to a variety of pathogens and help in clearance [ 9 , 15 ]. Recent evidences indicate that in addition to their pathogen recognition property, SP-A and SP-D also play an important role in stimulating immuno-regulatory pathways [ 15 ]. However, the collectins' role in coccidioidomycosis is not known. This study focuses on analyzing the changes in amounts of the SP-A and SP-D in the bronchoalveolar lavage fluid (BALF) samples from mice infected with lethal dose of  C. posadasii  and  C. posadasii  protected mice after immunization with protective formalin killed spherule (FKS) vaccine, and binding of pulmonary collectins to Coccidioidal antigens.
Background
Coccidioidomycosis or Valley Fever is a fungal disease caused by the biphasic, highly virulent, soil-fungus  Coccidioides immitis  or  posadasii  [ 1 ]. It is endemic in the southwest regions of US, Northern Mexico and parts of Central America [ 2 ].  C. posadasii or C. immitis , are the most virulent fungal pathogens enlisted in Select Agent list and pose a risk for bioterrorism [ 3 ]. The primary infection is acquired by inhalation of air-borne, mycelial phase arthroconidium that converts into endosporulating spherule in the lung. Clinical manifestations of the disease range from pulmonary infection to a more severe fatal mycosis involving extra-pulmonary tissues in 1–10% of the infected people [ 1 - 4 ]. Previous studies suggest that Th1 cell mediated immunity protects individuals against  Coccidioides  [ 5 , 6 ]. However, information is lacking regarding the pulmonary innate immune components that may play a critical role in regulation of immune responses against  Coccidioides .Coccidioides immitisposadasiiC. posadasii or C. immitisCoccidioidesCoccidioidesAt alveolar level in the lung, the innate immune system is composed of many cell types and chemical mediators, including surfactant. The pulmonary surfactant is a complex mixture of lipids (88–90%) and proteins (10–12%), synthesized by type II epithelial cells and Clara cells. It lines the alveoli, and helps in maintaining normal lung function [ 7 ]. Among four different surfactant proteins, surfactant proteins-A (SP-A) and D (SP-D) are members of the "Collectin" family [ 8 ]. In the past, several studies have suggested that both SP-A and SP-D play an important role in innate host defense against various viral, fungal and bacterial pathogens [ 9 , 10 ]. More evidence for the pulmonary collectins' role in host defense comes from studies on SP-A- deficient mice that are susceptible to intra-tracheal Group B  Streptococci  [ 11 ],  Pseudomonas aeruginosa  [ 12 ], and Respiratory Syncytial Virus [ 13 ]. Also, intranasally administered SP-D has been found to reduce replication of Respiratory Syncytial Virus in the lungs of infected mice [ 14 ]. Both SP-A and SP-D, have been classified as secretory pattern-recognition receptors that can bind to a variety of pathogens and help in clearance [ 9 , 15 ]. Recent evidences indicate that in addition to their pathogen recognition property, SP-A and SP-D also play an important role in stimulating immuno-regulatory pathways [ 15 ]. However, the collectins' role in coccidioidomycosis is not known.StreptococciPseudomonas aeruginosaThis study focuses on analyzing the changes in amounts of the SP-A and SP-D in the bronchoalveolar lavage fluid (BALF) samples from mice infected with lethal dose of  C. posadasii  and  C. posadasii  protected mice after immunization with protective formalin killed spherule (FKS) vaccine, and binding of pulmonary collectins to Coccidioidal antigens.C. posadasiiC. posadasiiMethods Mice BALB/c and C57BL6 mice (6 weeks old female) from Jackson Laboratory (Bar Harbour, ME) were used in this study. Both mouse strains are susceptible to  C. posadasii  infection. The BALB/c mice were used to study the changes in pulmonary surfactant after intranasal challenge with  C. posadasii . And, the C57BL6 mice were used to study the changes in pulmonary surfactant after vaccination with protective FKS vaccine. Mice were housed in Biosafety Level-3 animal facility at UTHSCSA and provided with food and water ad libitum. All experimental animal care and treatment protocols were reviewed and approved by Institutional Animal Care and Use Committee. Coccidioides posadasii C. immitis  (now  posadasii ) Silveira strain, cultured on 1 % glucose-0.5 % yeast extract agar (GYE), was used for infecting the mice [ 1 ]. The arthroconidia were harvested in endotoxin-free 0.15 M saline (Baxter Health Care Products, Deerfield, IL) from 6–8 weeks old mycelial phase cultures grown on GYE plates. The arthroconidia suspension was passed over a sterile cotton column to remove hyphal elements and arthroconidia were enumerated by hemacytometer counts. The viable cfu counts were confirmed pre- and post-infection by plate cultures on GYE agar. All the experiments with  C. posadasii  were carried out in Biosafety Level-3 facility at UTHSCSA. Intranasal Challenge with  C. posadasii  Arthroconidia Mice were anaesthetized after intramuscular injection of ketamine- xylazine (75 μg/g body weight ketamine and 10 μg/g body weight xylazine) and were then challenged intranasally with a lethal dose of arthroconidia (n = 30, fresh harvest of  C. posadasii  arthroconidia) suspended in endotoxin-free 0.15 M NaCl (Baxter Health Care Corp, Deerfield, IL) using sterile pyrogen-free microtip. Mice were held in an upright position for 1–2 min to resume normal breathing after injection. Control mice were challenged with equal volume of endotoxin-free 0.15 M NaCl. Preparation of  Coccidioide -FKS vaccine and Immunization of Mice C. posadasii  (strain  Silveira )arthroconidia were used to prepare FKS as described earlier [ 16 ]. Briefly, arthroconidia were inoculated in modified Converse medium containing Tamol and cultured while shaking at 180 rpm at 40°C in 20 % CO 2  incubator. The spherules were collected from the harvested culture, washed in endotoxin-free water and killed with 1 % formalin. FKS preparation was checked for sterility and lyophilized. C57BL6 female mice (age 6 weeks old) were immunized intramuscularly twice and subcutaneously once at one week interval with FKS (0.7 mg/dose each time). The mice in FKS immunized, infected group were then challenged intranasally with 30  C. posadasii  arthroconidia, 15 days after last immunization, as described above. Fungal Burden Assay Mice were anaesthetized as mentioned above, prior to sacrifice on day 10, post intranasal infection. This standard procedure was used for intranasal injection since it does not cause respiratory depression during anaesthesia. The lung and spleen tissues were collected in sterile 0.15 M NaCl for studying fungal load. The fungal burden was studied by plating ten fold dilutions of lung and spleen homogenates in 0.15 M saline on Mycosel agar plates (BD Biosciences, Franklin Lakes, NJ) and incubating for 72 h at 30°C. The cfu counts were recorded and normalized with organ weight. Collection and Processing of BALF At the time of necropsy, we collected BALF by injecting 1 ml endotoxin-free 0.15 M NaCl solution (Baxter Health Care Corp, Deerfield, IL) three times, via an angiocatheter (BD Biosciences, San Diego, CA) placed in the trachea. The volume of the input solution was kept constant (3 ml total) and approximately, 90–95 % of the solution was recovered consistently. The BALF was centrifuged at 500 rpm for 10 min at 4°C to remove cells. The cell free BALF supernatant was filtered through 0.2 μm syringe filters (Nalge Nunc International, Rochester, NY) and stored at -80°C for further analysis. Total Protein and Lipid Analysis The total protein concentration was measured in BALF specimens using micro bicinchonic acid protein assay kit (Pierce, Rockford, IL) against bovine serum albumin (BSA) standard protein. The total phospholipid content in lipid extracts of BALF specimens was determined using the method of Stewart, against Dipalmitoyl-phosphatidylcholine (DPPC, Avanti Polar Lipids, Alabaster, AL) standard solutions [ 17 , 18 ]. Briefly, the lipid extract of BALF specimens and DPPC standard solutions was completely dried under compressed nitrogen gas. The dried lipids were dissolved in chloroform and mixed with 1 ml of 2.7% ferric chloride and 3% ammonium thiocyanate in glass tubes. The mixture was vortex mixed for 1 min and centrifuged at 200 rpm for 5 min. The bottom red lower layer of phospholipids and ammonium ferro-thiocyanate complex was collected and absorbance was read at 488 nm. Western Blotting The BALF and lung tissue homogenate samples (total protein 10–50 μg) were run on 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) running gel and transferred on nitrocellulose membranes (Schleicher & Schuell, Keene, NH) overnight at 15 mA current. The nonspecific sites on the membrane with transferred proteins were blocked by 15% nonfat milk in Tris-buffered saline containing 0.05% tween 20 (TBST). The membrane was washed and incubated for 1 h with diluted (1:500) primary anti-human SP-A polyclonal antibody raised in rabbit (obtained from Dr. Richard J. King, UTHSCSA, San Antonio, TX) or anti-mouse SP-D antibody (kindly provided by Dr. Jo Rae Wright, Duke University Medical Center, Durham, NC). After washing the membrane with TBST, the membrane was incubated for 1 h with 1:10,000 diluted alkaline phosphatase conjugated anti-rabbit IgG raised in goat (Sigma Chemical Co, St. Louis, MO). The immunoreactive bands were observed by alkaline phosphatase conjugate system (Biorad, Hercules, CA). Purified human SP-A (kindly provided by Dr. Richard J. King, UTHSCSA, San Antonio, TX) and recombinant human SP-D (kindly provided by Dr. Erika C. Crouch, Washington University in St. Louis, St. Louis, MO) were run with the samples. The Coccidioidal antigens: lysates and filtrates of Coccidioidin (CDN), prepared as a toluene-induced lysate of young  C. posadasii  mycelia (obtained from Dr. Rebecca A. Cox, UTHSCSA, San Antonio, TX, [ 19 ]) were also run to check the cross-reactivities of anti-SP-A and SP-D antibodies to fungal antigens. Enzyme-Linked Immunosorbent Assay (ELISA) for SP-A and SP-D The concentrations of SP-A and SP-D were measured in BALF samples as described earlier [ 20 ]. The antibodies against SP-A and SP-D reacted with 34 kDa (SP-A) and 43 kDa (SP-D) immunroreactive bands in BALF and lung tissue homogenates (Fig  1 ). For measuring the lavage concentrations of SP-A and SP-D, the indirect ELISA procedure was used [ 20 ]. Briefly, the wells of Immulon 4 strips (Dynatech, Chantilly, VA) were coated overnight with purified human SP-A or recombinant human SP-D antigens (standards) and diluted BALF (three different dilutions) in 0.1 M NaHCO 3 , pH 9.6. The wells were washed three times with deionized water, and nonspecific sites were blocked with a buffer containing 0.25% BSA, 0.05% tween 20, 0.17 M boric acid and 0.12 M NaCl, pH 8.5. The wells were washed and incubated for 2 h with rabbit anti-human SP-A or rabbit anti-mouse SP-D antibody. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent (Sigma Chemical Co. St. Louis, MO). The reaction was stopped by adding 50 μl of 2 N H 2 SO 4  and read at 450 nm spectrophotometrically. The regression coefficient for a least-square linear fit to the standard curve of SP-A and SP-D was 0.99. The limits of detection for SP-A and SP-D were 2 ng/ml. Figure 1 Western blot for (A) SP-A and (B) SP-D proteins in mouse lung. Lanes (a, b): 2.5 μg total lavage fluid protein (c, d): 100 μg of total lung tissue homogenate protein from two healthy, non-infected BALB/c mice, and (e): 10 ng purified human SP-A or recombinant SP-D protein. Binding of SP-A and SP-D to Coccidioidal Antigens A microtiter well based method [ 21 ] was used to study the SP-A and SP-D interactions with Coccidioidal antigens (CDN-lysate and CDN-filtrate). Briefly, microtiter wells (Immulon 4; Dynatech, Chantilly, VA) were coated with 50 μl of CDN-lysate (10 μg/ml diluted in 0.1 M NaHCO 3  buffer, pH 9.6) or CDN-filtrate (10 μg/ml diluted in 0.1 M NaHCO 3  buffer, pH 9.6) or BSA (10 μg/ml diluted in 0.1 M NaHCO 3  buffer, pH 9.6) at room temperature. The nonspecific binding was blocked with phosphate buffered saline (pH 7.4) containing 0.1% triton-X 100 and 3% nonfat milk (buffer A). The purified human SP-A and recombinant human SP-D diluted in 20 mM Tris (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl 2  and 1 mg/ml BSA were then added to the wells and incubated for 3 h at 37°C. The wells were then washed with buffer A and incubated for 1 h at room temperature with diluted (1:1000 in buffer A) anti-SP-A and anti-SP-D antibodies. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent (Sigma Chemical Co. St. Louis, MO). The reaction was stopped by adding 2 N H 2 SO 4  and read at 405 nm spectrophotometrically. The coating of Coccidioidal antigens (CDN-lysate and CDN-filtrate) to the plates was confirmed using a positive control antibody that recognizes Coccidioidal antigens as described earlier [ 22 ]. The alkaline phosphatase-conjugated rat anti-mouse IgG antibody (Zymed, San Francisco, CA) served as secondary detection antibody. Statistics Statistical analyses of the data (t-test or ANOVA) were done using Prism Software (Graphpad Software, San Diego, CA). The p value <0.05 was considered significant.
Methods
Mice BALB/c and C57BL6 mice (6 weeks old female) from Jackson Laboratory (Bar Harbour, ME) were used in this study. Both mouse strains are susceptible to  C. posadasii  infection. The BALB/c mice were used to study the changes in pulmonary surfactant after intranasal challenge with  C. posadasii . And, the C57BL6 mice were used to study the changes in pulmonary surfactant after vaccination with protective FKS vaccine. Mice were housed in Biosafety Level-3 animal facility at UTHSCSA and provided with food and water ad libitum. All experimental animal care and treatment protocols were reviewed and approved by Institutional Animal Care and Use Committee. Coccidioides posadasii C. immitis  (now  posadasii ) Silveira strain, cultured on 1 % glucose-0.5 % yeast extract agar (GYE), was used for infecting the mice [ 1 ]. The arthroconidia were harvested in endotoxin-free 0.15 M saline (Baxter Health Care Products, Deerfield, IL) from 6–8 weeks old mycelial phase cultures grown on GYE plates. The arthroconidia suspension was passed over a sterile cotton column to remove hyphal elements and arthroconidia were enumerated by hemacytometer counts. The viable cfu counts were confirmed pre- and post-infection by plate cultures on GYE agar. All the experiments with  C. posadasii  were carried out in Biosafety Level-3 facility at UTHSCSA.
Mice
BALB/c and C57BL6 mice (6 weeks old female) from Jackson Laboratory (Bar Harbour, ME) were used in this study. Both mouse strains are susceptible to  C. posadasii  infection. The BALB/c mice were used to study the changes in pulmonary surfactant after intranasal challenge with  C. posadasii . And, the C57BL6 mice were used to study the changes in pulmonary surfactant after vaccination with protective FKS vaccine. Mice were housed in Biosafety Level-3 animal facility at UTHSCSA and provided with food and water ad libitum. All experimental animal care and treatment protocols were reviewed and approved by Institutional Animal Care and Use Committee.C. posadasiiC. posadasiiCoccidioides posadasii C. immitis  (now  posadasii ) Silveira strain, cultured on 1 % glucose-0.5 % yeast extract agar (GYE), was used for infecting the mice [ 1 ]. The arthroconidia were harvested in endotoxin-free 0.15 M saline (Baxter Health Care Products, Deerfield, IL) from 6–8 weeks old mycelial phase cultures grown on GYE plates. The arthroconidia suspension was passed over a sterile cotton column to remove hyphal elements and arthroconidia were enumerated by hemacytometer counts. The viable cfu counts were confirmed pre- and post-infection by plate cultures on GYE agar. All the experiments with  C. posadasii  were carried out in Biosafety Level-3 facility at UTHSCSA.
Coccidioides posadasii
C. immitis  (now  posadasii ) Silveira strain, cultured on 1 % glucose-0.5 % yeast extract agar (GYE), was used for infecting the mice [ 1 ]. The arthroconidia were harvested in endotoxin-free 0.15 M saline (Baxter Health Care Products, Deerfield, IL) from 6–8 weeks old mycelial phase cultures grown on GYE plates. The arthroconidia suspension was passed over a sterile cotton column to remove hyphal elements and arthroconidia were enumerated by hemacytometer counts. The viable cfu counts were confirmed pre- and post-infection by plate cultures on GYE agar. All the experiments with  C. posadasii  were carried out in Biosafety Level-3 facility at UTHSCSA.C. immitisposadasiiC. posadasiiIntranasal Challenge with  C. posadasii  Arthroconidia Mice were anaesthetized after intramuscular injection of ketamine- xylazine (75 μg/g body weight ketamine and 10 μg/g body weight xylazine) and were then challenged intranasally with a lethal dose of arthroconidia (n = 30, fresh harvest of  C. posadasii  arthroconidia) suspended in endotoxin-free 0.15 M NaCl (Baxter Health Care Corp, Deerfield, IL) using sterile pyrogen-free microtip. Mice were held in an upright position for 1–2 min to resume normal breathing after injection. Control mice were challenged with equal volume of endotoxin-free 0.15 M NaCl.
Intranasal Challenge with  C. posadasii  Arthroconidia
C. posadasiiMice were anaesthetized after intramuscular injection of ketamine- xylazine (75 μg/g body weight ketamine and 10 μg/g body weight xylazine) and were then challenged intranasally with a lethal dose of arthroconidia (n = 30, fresh harvest of  C. posadasii  arthroconidia) suspended in endotoxin-free 0.15 M NaCl (Baxter Health Care Corp, Deerfield, IL) using sterile pyrogen-free microtip. Mice were held in an upright position for 1–2 min to resume normal breathing after injection. Control mice were challenged with equal volume of endotoxin-free 0.15 M NaCl.C. posadasiiPreparation of  Coccidioide -FKS vaccine and Immunization of Mice C. posadasii  (strain  Silveira )arthroconidia were used to prepare FKS as described earlier [ 16 ]. Briefly, arthroconidia were inoculated in modified Converse medium containing Tamol and cultured while shaking at 180 rpm at 40°C in 20 % CO 2  incubator. The spherules were collected from the harvested culture, washed in endotoxin-free water and killed with 1 % formalin. FKS preparation was checked for sterility and lyophilized. C57BL6 female mice (age 6 weeks old) were immunized intramuscularly twice and subcutaneously once at one week interval with FKS (0.7 mg/dose each time). The mice in FKS immunized, infected group were then challenged intranasally with 30  C. posadasii  arthroconidia, 15 days after last immunization, as described above.
Preparation of  Coccidioide -FKS vaccine and Immunization of Mice
CoccidioideC. posadasii  (strain  Silveira )arthroconidia were used to prepare FKS as described earlier [ 16 ]. Briefly, arthroconidia were inoculated in modified Converse medium containing Tamol and cultured while shaking at 180 rpm at 40°C in 20 % CO 2  incubator. The spherules were collected from the harvested culture, washed in endotoxin-free water and killed with 1 % formalin. FKS preparation was checked for sterility and lyophilized. C57BL6 female mice (age 6 weeks old) were immunized intramuscularly twice and subcutaneously once at one week interval with FKS (0.7 mg/dose each time). The mice in FKS immunized, infected group were then challenged intranasally with 30  C. posadasii  arthroconidia, 15 days after last immunization, as described above.C. posadasiiSilveiraC. posadasiiFungal Burden Assay Mice were anaesthetized as mentioned above, prior to sacrifice on day 10, post intranasal infection. This standard procedure was used for intranasal injection since it does not cause respiratory depression during anaesthesia. The lung and spleen tissues were collected in sterile 0.15 M NaCl for studying fungal load. The fungal burden was studied by plating ten fold dilutions of lung and spleen homogenates in 0.15 M saline on Mycosel agar plates (BD Biosciences, Franklin Lakes, NJ) and incubating for 72 h at 30°C. The cfu counts were recorded and normalized with organ weight.
Fungal Burden Assay
Mice were anaesthetized as mentioned above, prior to sacrifice on day 10, post intranasal infection. This standard procedure was used for intranasal injection since it does not cause respiratory depression during anaesthesia. The lung and spleen tissues were collected in sterile 0.15 M NaCl for studying fungal load.The fungal burden was studied by plating ten fold dilutions of lung and spleen homogenates in 0.15 M saline on Mycosel agar plates (BD Biosciences, Franklin Lakes, NJ) and incubating for 72 h at 30°C. The cfu counts were recorded and normalized with organ weight.Collection and Processing of BALF At the time of necropsy, we collected BALF by injecting 1 ml endotoxin-free 0.15 M NaCl solution (Baxter Health Care Corp, Deerfield, IL) three times, via an angiocatheter (BD Biosciences, San Diego, CA) placed in the trachea. The volume of the input solution was kept constant (3 ml total) and approximately, 90–95 % of the solution was recovered consistently. The BALF was centrifuged at 500 rpm for 10 min at 4°C to remove cells. The cell free BALF supernatant was filtered through 0.2 μm syringe filters (Nalge Nunc International, Rochester, NY) and stored at -80°C for further analysis.
Collection and Processing of BALF
At the time of necropsy, we collected BALF by injecting 1 ml endotoxin-free 0.15 M NaCl solution (Baxter Health Care Corp, Deerfield, IL) three times, via an angiocatheter (BD Biosciences, San Diego, CA) placed in the trachea. The volume of the input solution was kept constant (3 ml total) and approximately, 90–95 % of the solution was recovered consistently. The BALF was centrifuged at 500 rpm for 10 min at 4°C to remove cells. The cell free BALF supernatant was filtered through 0.2 μm syringe filters (Nalge Nunc International, Rochester, NY) and stored at -80°C for further analysis.Total Protein and Lipid Analysis The total protein concentration was measured in BALF specimens using micro bicinchonic acid protein assay kit (Pierce, Rockford, IL) against bovine serum albumin (BSA) standard protein. The total phospholipid content in lipid extracts of BALF specimens was determined using the method of Stewart, against Dipalmitoyl-phosphatidylcholine (DPPC, Avanti Polar Lipids, Alabaster, AL) standard solutions [ 17 , 18 ]. Briefly, the lipid extract of BALF specimens and DPPC standard solutions was completely dried under compressed nitrogen gas. The dried lipids were dissolved in chloroform and mixed with 1 ml of 2.7% ferric chloride and 3% ammonium thiocyanate in glass tubes. The mixture was vortex mixed for 1 min and centrifuged at 200 rpm for 5 min. The bottom red lower layer of phospholipids and ammonium ferro-thiocyanate complex was collected and absorbance was read at 488 nm.
Total Protein and Lipid Analysis
The total protein concentration was measured in BALF specimens using micro bicinchonic acid protein assay kit (Pierce, Rockford, IL) against bovine serum albumin (BSA) standard protein. The total phospholipid content in lipid extracts of BALF specimens was determined using the method of Stewart, against Dipalmitoyl-phosphatidylcholine (DPPC, Avanti Polar Lipids, Alabaster, AL) standard solutions [ 17 , 18 ]. Briefly, the lipid extract of BALF specimens and DPPC standard solutions was completely dried under compressed nitrogen gas. The dried lipids were dissolved in chloroform and mixed with 1 ml of 2.7% ferric chloride and 3% ammonium thiocyanate in glass tubes. The mixture was vortex mixed for 1 min and centrifuged at 200 rpm for 5 min. The bottom red lower layer of phospholipids and ammonium ferro-thiocyanate complex was collected and absorbance was read at 488 nm.Western Blotting The BALF and lung tissue homogenate samples (total protein 10–50 μg) were run on 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) running gel and transferred on nitrocellulose membranes (Schleicher & Schuell, Keene, NH) overnight at 15 mA current. The nonspecific sites on the membrane with transferred proteins were blocked by 15% nonfat milk in Tris-buffered saline containing 0.05% tween 20 (TBST). The membrane was washed and incubated for 1 h with diluted (1:500) primary anti-human SP-A polyclonal antibody raised in rabbit (obtained from Dr. Richard J. King, UTHSCSA, San Antonio, TX) or anti-mouse SP-D antibody (kindly provided by Dr. Jo Rae Wright, Duke University Medical Center, Durham, NC). After washing the membrane with TBST, the membrane was incubated for 1 h with 1:10,000 diluted alkaline phosphatase conjugated anti-rabbit IgG raised in goat (Sigma Chemical Co, St. Louis, MO). The immunoreactive bands were observed by alkaline phosphatase conjugate system (Biorad, Hercules, CA). Purified human SP-A (kindly provided by Dr. Richard J. King, UTHSCSA, San Antonio, TX) and recombinant human SP-D (kindly provided by Dr. Erika C. Crouch, Washington University in St. Louis, St. Louis, MO) were run with the samples. The Coccidioidal antigens: lysates and filtrates of Coccidioidin (CDN), prepared as a toluene-induced lysate of young  C. posadasii  mycelia (obtained from Dr. Rebecca A. Cox, UTHSCSA, San Antonio, TX, [ 19 ]) were also run to check the cross-reactivities of anti-SP-A and SP-D antibodies to fungal antigens.
Western Blotting
The BALF and lung tissue homogenate samples (total protein 10–50 μg) were run on 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) running gel and transferred on nitrocellulose membranes (Schleicher & Schuell, Keene, NH) overnight at 15 mA current. The nonspecific sites on the membrane with transferred proteins were blocked by 15% nonfat milk in Tris-buffered saline containing 0.05% tween 20 (TBST). The membrane was washed and incubated for 1 h with diluted (1:500) primary anti-human SP-A polyclonal antibody raised in rabbit (obtained from Dr. Richard J. King, UTHSCSA, San Antonio, TX) or anti-mouse SP-D antibody (kindly provided by Dr. Jo Rae Wright, Duke University Medical Center, Durham, NC). After washing the membrane with TBST, the membrane was incubated for 1 h with 1:10,000 diluted alkaline phosphatase conjugated anti-rabbit IgG raised in goat (Sigma Chemical Co, St. Louis, MO). The immunoreactive bands were observed by alkaline phosphatase conjugate system (Biorad, Hercules, CA). Purified human SP-A (kindly provided by Dr. Richard J. King, UTHSCSA, San Antonio, TX) and recombinant human SP-D (kindly provided by Dr. Erika C. Crouch, Washington University in St. Louis, St. Louis, MO) were run with the samples.The Coccidioidal antigens: lysates and filtrates of Coccidioidin (CDN), prepared as a toluene-induced lysate of young  C. posadasii  mycelia (obtained from Dr. Rebecca A. Cox, UTHSCSA, San Antonio, TX, [ 19 ]) were also run to check the cross-reactivities of anti-SP-A and SP-D antibodies to fungal antigens.C. posadasiiEnzyme-Linked Immunosorbent Assay (ELISA) for SP-A and SP-D The concentrations of SP-A and SP-D were measured in BALF samples as described earlier [ 20 ]. The antibodies against SP-A and SP-D reacted with 34 kDa (SP-A) and 43 kDa (SP-D) immunroreactive bands in BALF and lung tissue homogenates (Fig  1 ). For measuring the lavage concentrations of SP-A and SP-D, the indirect ELISA procedure was used [ 20 ]. Briefly, the wells of Immulon 4 strips (Dynatech, Chantilly, VA) were coated overnight with purified human SP-A or recombinant human SP-D antigens (standards) and diluted BALF (three different dilutions) in 0.1 M NaHCO 3 , pH 9.6. The wells were washed three times with deionized water, and nonspecific sites were blocked with a buffer containing 0.25% BSA, 0.05% tween 20, 0.17 M boric acid and 0.12 M NaCl, pH 8.5. The wells were washed and incubated for 2 h with rabbit anti-human SP-A or rabbit anti-mouse SP-D antibody. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent (Sigma Chemical Co. St. Louis, MO). The reaction was stopped by adding 50 μl of 2 N H 2 SO 4  and read at 450 nm spectrophotometrically. The regression coefficient for a least-square linear fit to the standard curve of SP-A and SP-D was 0.99. The limits of detection for SP-A and SP-D were 2 ng/ml. Figure 1 Western blot for (A) SP-A and (B) SP-D proteins in mouse lung. Lanes (a, b): 2.5 μg total lavage fluid protein (c, d): 100 μg of total lung tissue homogenate protein from two healthy, non-infected BALB/c mice, and (e): 10 ng purified human SP-A or recombinant SP-D protein.
Enzyme-Linked Immunosorbent Assay (ELISA) for SP-A and SP-D
The concentrations of SP-A and SP-D were measured in BALF samples as described earlier [ 20 ]. The antibodies against SP-A and SP-D reacted with 34 kDa (SP-A) and 43 kDa (SP-D) immunroreactive bands in BALF and lung tissue homogenates (Fig  1 ). For measuring the lavage concentrations of SP-A and SP-D, the indirect ELISA procedure was used [ 20 ]. Briefly, the wells of Immulon 4 strips (Dynatech, Chantilly, VA) were coated overnight with purified human SP-A or recombinant human SP-D antigens (standards) and diluted BALF (three different dilutions) in 0.1 M NaHCO 3 , pH 9.6. The wells were washed three times with deionized water, and nonspecific sites were blocked with a buffer containing 0.25% BSA, 0.05% tween 20, 0.17 M boric acid and 0.12 M NaCl, pH 8.5. The wells were washed and incubated for 2 h with rabbit anti-human SP-A or rabbit anti-mouse SP-D antibody. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent (Sigma Chemical Co. St. Louis, MO). The reaction was stopped by adding 50 μl of 2 N H 2 SO 4  and read at 450 nm spectrophotometrically. The regression coefficient for a least-square linear fit to the standard curve of SP-A and SP-D was 0.99. The limits of detection for SP-A and SP-D were 2 ng/ml.Binding of SP-A and SP-D to Coccidioidal Antigens A microtiter well based method [ 21 ] was used to study the SP-A and SP-D interactions with Coccidioidal antigens (CDN-lysate and CDN-filtrate). Briefly, microtiter wells (Immulon 4; Dynatech, Chantilly, VA) were coated with 50 μl of CDN-lysate (10 μg/ml diluted in 0.1 M NaHCO 3  buffer, pH 9.6) or CDN-filtrate (10 μg/ml diluted in 0.1 M NaHCO 3  buffer, pH 9.6) or BSA (10 μg/ml diluted in 0.1 M NaHCO 3  buffer, pH 9.6) at room temperature. The nonspecific binding was blocked with phosphate buffered saline (pH 7.4) containing 0.1% triton-X 100 and 3% nonfat milk (buffer A). The purified human SP-A and recombinant human SP-D diluted in 20 mM Tris (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl 2  and 1 mg/ml BSA were then added to the wells and incubated for 3 h at 37°C. The wells were then washed with buffer A and incubated for 1 h at room temperature with diluted (1:1000 in buffer A) anti-SP-A and anti-SP-D antibodies. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent (Sigma Chemical Co. St. Louis, MO). The reaction was stopped by adding 2 N H 2 SO 4  and read at 405 nm spectrophotometrically. The coating of Coccidioidal antigens (CDN-lysate and CDN-filtrate) to the plates was confirmed using a positive control antibody that recognizes Coccidioidal antigens as described earlier [ 22 ]. The alkaline phosphatase-conjugated rat anti-mouse IgG antibody (Zymed, San Francisco, CA) served as secondary detection antibody.
Binding of SP-A and SP-D to Coccidioidal Antigens
A microtiter well based method [ 21 ] was used to study the SP-A and SP-D interactions with Coccidioidal antigens (CDN-lysate and CDN-filtrate). Briefly, microtiter wells (Immulon 4; Dynatech, Chantilly, VA) were coated with 50 μl of CDN-lysate (10 μg/ml diluted in 0.1 M NaHCO 3  buffer, pH 9.6) or CDN-filtrate (10 μg/ml diluted in 0.1 M NaHCO 3  buffer, pH 9.6) or BSA (10 μg/ml diluted in 0.1 M NaHCO 3  buffer, pH 9.6) at room temperature. The nonspecific binding was blocked with phosphate buffered saline (pH 7.4) containing 0.1% triton-X 100 and 3% nonfat milk (buffer A). The purified human SP-A and recombinant human SP-D diluted in 20 mM Tris (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl 2  and 1 mg/ml BSA were then added to the wells and incubated for 3 h at 37°C. The wells were then washed with buffer A and incubated for 1 h at room temperature with diluted (1:1000 in buffer A) anti-SP-A and anti-SP-D antibodies. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent (Sigma Chemical Co. St. Louis, MO). The reaction was stopped by adding 2 N H 2 SO 4  and read at 405 nm spectrophotometrically.The coating of Coccidioidal antigens (CDN-lysate and CDN-filtrate) to the plates was confirmed using a positive control antibody that recognizes Coccidioidal antigens as described earlier [ 22 ]. The alkaline phosphatase-conjugated rat anti-mouse IgG antibody (Zymed, San Francisco, CA) served as secondary detection antibody.Statistics Statistical analyses of the data (t-test or ANOVA) were done using Prism Software (Graphpad Software, San Diego, CA). The p value <0.05 was considered significant.
Statistics
Statistical analyses of the data (t-test or ANOVA) were done using Prism Software (Graphpad Software, San Diego, CA). The p value <0.05 was considered significant.Results Pathological status All of the  C. posadasii  infected mice survived till the day of sacrifice (day 10 post infection). However, the mice were lethargic and lost body weight (Table  1 ). Abscess like lesions were quite evident on gross examination of the lung. The total wet lung weights were increased in  C. posadasii  infected mice. Table 1 Body weights (g) of  C. posadasii  infected and non-infected BALB/c mice (n = 10 of each type). Values are shown as Mean (SEM) of one representative experiment of two independent experiments. Days post challenge → Mice ↓ Day 0 Day 10 Non-infected 18.36 (0.29) 19.46 (0.23) ** C. posadasii  infected 17.77 (0.39) 16.35 (0.53) *, # ** p < 0.01 as compared to non-infected control mice at day 0. *p < 0.05, # p < 0.0001 as compared to  C. posadasii  infected mice at day 0 and non-infected mice at day 10, respectively (t-test). The mean protein content of BALF samples from infected mice was 788 μg versus 326 μg protein in BALF samples from non-infected saline injected control mice, after 10 days of intranasal infection (p < 0.05, Table  2 ). In contrast, the phospholipid concentration was reduced in BALF samples from  C. posadasii  infected mice (58 μg) when compared to non-infected saline injected controls (165 μg, p < 0.05). Table 2 Total protein and phospholipid contents in BALF samples from non-infected and  C. posadasii  infected BALB/c mice (n = 5 of each type). Values are shown as Mean (SEM) from one represenative experiment of two independent experiments. Mice Total protein (μg) Total phospholipid (μg) Non-infected 326.0 (26.9) 165.5 (10.8) C. posadasii  infected 788.7 (248.6)* 58.7 (15.7)* * p < 0.05 as compared to non-infected control mice (t-test). The amounts of SP-A, SP-D and phospholipid are reduced in BALF samples from  C. posadasii  infected mice The anti-human-SP-A and anti-mouse SP-D antibodies recognized 34 kDa and 43 kDa monomer bands of SP-A and SP-D in BALF and lung tissue samples (Fig  1 ). The upper bands of approximately 68 kDa and 85 kDa size (dimer of SP-A and SP-D protein) were also visible in lanes e of Figure  1A  and  1B  due to incomplete reduction, respectively. There were no differences in the detectable isoforms of SP-A or SP-D in the BALF samples from infected mice as compared to non-infected control mice (data not shown). The antibodies did not cross-react with Coccidioidal antigens in CDN lysate or CDN filtrate (Fig  2 ). The amounts of SP-A and SP-D were significantly reduced in BALF samples from  C. posadasii  infected BALB/c mice when compared to saline injected, non-infected control mice after 10 days of intranasal challenge (p < 0.001) (Fig  3A ). No significant changes were observed in the amounts of SP-A and SP-D in BALF samples collected from BALB/c mice, 5 days after intranasal challenge with  C. posadasii  (data not shown). Figure 2 Western blot of CDN-lysate and CDN-filtrate for crossreactivity with (A) anti-human SP-A and (b) anti-mouse SP-D antibodies. Lanes (a): 20 μg, (b): 10 μg and (c): 1 μg CDN-filtrate protein. Lanes (d): 20 μg, (e): 10 μg and (f): 1 μg CDN-lysate protein and last lane: 10 ng purified human SP-A protein or recombinant SP-D protein. Figure 3 SP-A and SP-D levels in BALF samples from (A)  C. posadasii  infected BALB/c mice (ng/μg protein, % of non-infected control mice, n = 5 of each type) (B) FKS immunized,  C. posadasii  infected C57BL6 mice (protected mice) (ng/μg protein, % of FKS immunized non-infected mice, n = 5 of each type). The data are shown from one representative experiment of two independent experiments. * p < 0.001 (ANOVA) The fungal colonies of  C. posadasii  were recovered from both lung and spleens of infected animals indicating the presence of active infection (Fig  4 ). Recovery of fungus in the spleen provides evidence of dissemination of  C. posadasii to  extra-pulmonary organs. Figure 4 Fungal load in the lung and spleen tissues of  C. posadasii  infected BALB/c mice (n = 5). The numbers of fungal colonies (CFU) were normalized with organ weight (g). Lavage SP-A and SP-D levels are unaltered in  C. posadasii  protected mice No significant changes were seen in SP-A or SP-D levels in BALF samples of protected (FKS immunized,  C. posadasii  arthroconidia infected) C57BL6 mice when compared to FKS immunized, non-infected mice (Fig  3B ). Also, there was no significant change in total protein content in BALF samples of FKS immunized,  C. posadasii  arthroconidia infected mice (890.9 μg) versus FKS immunized, non-infected mice (538.4 μg). SP-A and SP-D bind to Coccidioidal antigens We further examined the binding of SP-A and SP-D to Coccidioidal antigens (CDN-lysate and CDN-filtrate) coated onto microtiter wells (Fig  5 ). Both SP-A and SP-D bound to coccidioidal antigens, but not to BSA in a concentration-dependent manner (Fig  5 ). Binding of SP-A to CDN-lysate and CDN-filtrate antigens was saturable, and maximum SP-A binding was reached between 2.5–5 μg/ml and 5–10 μg/ml, respectively (Fig  5A ). Similarly binding of SP-D to CDN-lysate and CDN-filtrate antigens was also saturable, and maximum SP-D binding was reached between 5–10 μg/ml (Fig  5B ). Figure 5 Binding of (A) SP-A and (B) SP-D to Coccidioidal antigens (CDN-lysate and CDN-filtrate). The binding of 1–10 μg/ml purified human SP-A or recombinant SP-D proteins was detected in CDN-lysate or CDN-filtrate or BSA coated wells (0.5 μg/well). Results are from one representative experiment of two independent experiments performed in duplicate. Values are shown as mean+SEM. In some cases, the error bars are smaller than the symbols.
Results
Pathological status All of the  C. posadasii  infected mice survived till the day of sacrifice (day 10 post infection). However, the mice were lethargic and lost body weight (Table  1 ). Abscess like lesions were quite evident on gross examination of the lung. The total wet lung weights were increased in  C. posadasii  infected mice. Table 1 Body weights (g) of  C. posadasii  infected and non-infected BALB/c mice (n = 10 of each type). Values are shown as Mean (SEM) of one representative experiment of two independent experiments. Days post challenge → Mice ↓ Day 0 Day 10 Non-infected 18.36 (0.29) 19.46 (0.23) ** C. posadasii  infected 17.77 (0.39) 16.35 (0.53) *, # ** p < 0.01 as compared to non-infected control mice at day 0. *p < 0.05, # p < 0.0001 as compared to  C. posadasii  infected mice at day 0 and non-infected mice at day 10, respectively (t-test). The mean protein content of BALF samples from infected mice was 788 μg versus 326 μg protein in BALF samples from non-infected saline injected control mice, after 10 days of intranasal infection (p < 0.05, Table  2 ). In contrast, the phospholipid concentration was reduced in BALF samples from  C. posadasii  infected mice (58 μg) when compared to non-infected saline injected controls (165 μg, p < 0.05). Table 2 Total protein and phospholipid contents in BALF samples from non-infected and  C. posadasii  infected BALB/c mice (n = 5 of each type). Values are shown as Mean (SEM) from one represenative experiment of two independent experiments. Mice Total protein (μg) Total phospholipid (μg) Non-infected 326.0 (26.9) 165.5 (10.8) C. posadasii  infected 788.7 (248.6)* 58.7 (15.7)* * p < 0.05 as compared to non-infected control mice (t-test).
Pathological status
All of the  C. posadasii  infected mice survived till the day of sacrifice (day 10 post infection). However, the mice were lethargic and lost body weight (Table  1 ). Abscess like lesions were quite evident on gross examination of the lung. The total wet lung weights were increased in  C. posadasii  infected mice.C. posadasiiC. posadasiiBody weights (g) of  C. posadasii  infected and non-infected BALB/c mice (n = 10 of each type). Values are shown as Mean (SEM) of one representative experiment of two independent experiments.C. posadasiiDays post challenge → Mice ↓Day 0Day 10C. posadasii** p < 0.01 as compared to non-infected control mice at day 0.*p < 0.05, # p < 0.0001 as compared to  C. posadasii  infected mice at day 0 and non-infected mice at day 10, respectively (t-test).C. posadasiiThe mean protein content of BALF samples from infected mice was 788 μg versus 326 μg protein in BALF samples from non-infected saline injected control mice, after 10 days of intranasal infection (p < 0.05, Table  2 ). In contrast, the phospholipid concentration was reduced in BALF samples from  C. posadasii  infected mice (58 μg) when compared to non-infected saline injected controls (165 μg, p < 0.05).C. posadasiiTotal protein and phospholipid contents in BALF samples from non-infected and  C. posadasii  infected BALB/c mice (n = 5 of each type). Values are shown as Mean (SEM) from one represenative experiment of two independent experiments.C. posadasiiMiceTotal protein (μg)Total phospholipid (μg)C. posadasii* p < 0.05 as compared to non-infected control mice (t-test).The amounts of SP-A, SP-D and phospholipid are reduced in BALF samples from  C. posadasii  infected mice The anti-human-SP-A and anti-mouse SP-D antibodies recognized 34 kDa and 43 kDa monomer bands of SP-A and SP-D in BALF and lung tissue samples (Fig  1 ). The upper bands of approximately 68 kDa and 85 kDa size (dimer of SP-A and SP-D protein) were also visible in lanes e of Figure  1A  and  1B  due to incomplete reduction, respectively. There were no differences in the detectable isoforms of SP-A or SP-D in the BALF samples from infected mice as compared to non-infected control mice (data not shown). The antibodies did not cross-react with Coccidioidal antigens in CDN lysate or CDN filtrate (Fig  2 ). The amounts of SP-A and SP-D were significantly reduced in BALF samples from  C. posadasii  infected BALB/c mice when compared to saline injected, non-infected control mice after 10 days of intranasal challenge (p < 0.001) (Fig  3A ). No significant changes were observed in the amounts of SP-A and SP-D in BALF samples collected from BALB/c mice, 5 days after intranasal challenge with  C. posadasii  (data not shown). Figure 2 Western blot of CDN-lysate and CDN-filtrate for crossreactivity with (A) anti-human SP-A and (b) anti-mouse SP-D antibodies. Lanes (a): 20 μg, (b): 10 μg and (c): 1 μg CDN-filtrate protein. Lanes (d): 20 μg, (e): 10 μg and (f): 1 μg CDN-lysate protein and last lane: 10 ng purified human SP-A protein or recombinant SP-D protein. Figure 3 SP-A and SP-D levels in BALF samples from (A)  C. posadasii  infected BALB/c mice (ng/μg protein, % of non-infected control mice, n = 5 of each type) (B) FKS immunized,  C. posadasii  infected C57BL6 mice (protected mice) (ng/μg protein, % of FKS immunized non-infected mice, n = 5 of each type). The data are shown from one representative experiment of two independent experiments. * p < 0.001 (ANOVA) The fungal colonies of  C. posadasii  were recovered from both lung and spleens of infected animals indicating the presence of active infection (Fig  4 ). Recovery of fungus in the spleen provides evidence of dissemination of  C. posadasii to  extra-pulmonary organs. Figure 4 Fungal load in the lung and spleen tissues of  C. posadasii  infected BALB/c mice (n = 5). The numbers of fungal colonies (CFU) were normalized with organ weight (g).
The amounts of SP-A, SP-D and phospholipid are reduced in BALF samples from  C. posadasii  infected mice
C. posadasiiThe anti-human-SP-A and anti-mouse SP-D antibodies recognized 34 kDa and 43 kDa monomer bands of SP-A and SP-D in BALF and lung tissue samples (Fig  1 ). The upper bands of approximately 68 kDa and 85 kDa size (dimer of SP-A and SP-D protein) were also visible in lanes e of Figure  1A  and  1B  due to incomplete reduction, respectively. There were no differences in the detectable isoforms of SP-A or SP-D in the BALF samples from infected mice as compared to non-infected control mice (data not shown). The antibodies did not cross-react with Coccidioidal antigens in CDN lysate or CDN filtrate (Fig  2 ). The amounts of SP-A and SP-D were significantly reduced in BALF samples from  C. posadasii  infected BALB/c mice when compared to saline injected, non-infected control mice after 10 days of intranasal challenge (p < 0.001) (Fig  3A ). No significant changes were observed in the amounts of SP-A and SP-D in BALF samples collected from BALB/c mice, 5 days after intranasal challenge with  C. posadasii  (data not shown).C. posadasiiC. posadasiiThe fungal colonies of  C. posadasii  were recovered from both lung and spleens of infected animals indicating the presence of active infection (Fig  4 ). Recovery of fungus in the spleen provides evidence of dissemination of  C. posadasii to  extra-pulmonary organs.C. posadasiiC. posadasii toLavage SP-A and SP-D levels are unaltered in  C. posadasii  protected mice No significant changes were seen in SP-A or SP-D levels in BALF samples of protected (FKS immunized,  C. posadasii  arthroconidia infected) C57BL6 mice when compared to FKS immunized, non-infected mice (Fig  3B ). Also, there was no significant change in total protein content in BALF samples of FKS immunized,  C. posadasii  arthroconidia infected mice (890.9 μg) versus FKS immunized, non-infected mice (538.4 μg).
Lavage SP-A and SP-D levels are unaltered in  C. posadasii  protected mice
C. posadasiiNo significant changes were seen in SP-A or SP-D levels in BALF samples of protected (FKS immunized,  C. posadasii  arthroconidia infected) C57BL6 mice when compared to FKS immunized, non-infected mice (Fig  3B ). Also, there was no significant change in total protein content in BALF samples of FKS immunized,  C. posadasii  arthroconidia infected mice (890.9 μg) versus FKS immunized, non-infected mice (538.4 μg).C. posadasiiC. posadasiiSP-A and SP-D bind to Coccidioidal antigens We further examined the binding of SP-A and SP-D to Coccidioidal antigens (CDN-lysate and CDN-filtrate) coated onto microtiter wells (Fig  5 ). Both SP-A and SP-D bound to coccidioidal antigens, but not to BSA in a concentration-dependent manner (Fig  5 ). Binding of SP-A to CDN-lysate and CDN-filtrate antigens was saturable, and maximum SP-A binding was reached between 2.5–5 μg/ml and 5–10 μg/ml, respectively (Fig  5A ). Similarly binding of SP-D to CDN-lysate and CDN-filtrate antigens was also saturable, and maximum SP-D binding was reached between 5–10 μg/ml (Fig  5B ). Figure 5 Binding of (A) SP-A and (B) SP-D to Coccidioidal antigens (CDN-lysate and CDN-filtrate). The binding of 1–10 μg/ml purified human SP-A or recombinant SP-D proteins was detected in CDN-lysate or CDN-filtrate or BSA coated wells (0.5 μg/well). Results are from one representative experiment of two independent experiments performed in duplicate. Values are shown as mean+SEM. In some cases, the error bars are smaller than the symbols.
SP-A and SP-D bind to Coccidioidal antigens
We further examined the binding of SP-A and SP-D to Coccidioidal antigens (CDN-lysate and CDN-filtrate) coated onto microtiter wells (Fig  5 ). Both SP-A and SP-D bound to coccidioidal antigens, but not to BSA in a concentration-dependent manner (Fig  5 ). Binding of SP-A to CDN-lysate and CDN-filtrate antigens was saturable, and maximum SP-A binding was reached between 2.5–5 μg/ml and 5–10 μg/ml, respectively (Fig  5A ). Similarly binding of SP-D to CDN-lysate and CDN-filtrate antigens was also saturable, and maximum SP-D binding was reached between 5–10 μg/ml (Fig  5B ).Discussion In the present study we found that the levels of pulmonary surfactant collectins were altered in the lungs of  C. posadasii  infected mice, but were intact in lungs of  C. posadasii  protected mice after immunization with protective FKS vaccine. Furthermore, our results suggest that both SP-A and SP-D bind to Coccidioidal antigens. This is the first study where the amounts of SP-A and SP-D were measured in BALF samples from mice infected with lethal dose of  C. posadasii  and the binding of pulmonary collectins to Coccidioidal antigens was assessed. Since lung is the first organ of the body that comes into contact with air-borne  C. posadasii  arthroconidia, we hypothesized that the pulmonary surfactant may play an important role in regulating the immune response against  C. posadasii . Among four surfactant proteins, SP-A and SP-D, interact with most of the clinically important fungal pathogens including  Pneumocystis carinii  [ 23 ] Cryptococcus neoformans  [ 24 ],  Aspergillus fumigatus  [ 25 ] and  Candida albicans  [ 26 ]. SP-D has been shown to bind to  C. albicans  and directly inhibit growth by aggregation of the organism without involvement of macrophage dependent phagocytosis [ 27 ]. On the other hand, surfactant proteins also induce phagocytosis, activation and killing of  A. fumigatus  conidia and  C. neoformans  by alveolar macrophages and neutrophils [ 28 , 29 ]. In support of these findings, further evidence comes from a study by Madan et al., that suggests that the introduction of recombinant SP-D improves the lung function and increases the survival rate of mice infected with  A. fumigatus  [ 25 ]. The decrease in amounts of SP-A and SP-D during  C. posadasii  infection versus the unaltered amounts in the lungs of protected mice and binding of collectins to Coccidioidal antigens indicate that pulmonary collectins may be involved in uptake/phagocytosis of  C. posadasii  by antigen presenting cells and downstream immune regulation. Besides changes in amounts of SP-A and SP-D, a decrease in the amount of BALF phospholipids was also observed in  C. posadasii  infected mice (Table  2 ). Earlier, Sheehan et al., [ 29 ] and Hoffman et al., [ 30 ] have reported similar findings of reduced surfactant phospholipid level in BALF samples of rats and humans infected with  Pneumocystis carinii  [ 29 , 30 ]. To date, however, the information is lacking concerning how surfactant phospholipids may be involved in host defense [ 31 ]. Likewise, the mechanisms underlying the decrease of BALF surfactant in murine model of Coccidioidomycosis remain to be defined. We speculate that the reduction in the collectins and phospholipids could be either due to metabolic dysfunction of pulmonary type II epithelial cells during  C. posadasii  infection or due to their utilization in the binding and uptake of  C. posadasii  by local antigen presenting cells. The metabolic pathways of pulmonary type II epithelial cells may be affected by secondary inflammatory mediators, such as TNF-α or IL-1β, secreted by inflammatory cells during  C. posadasii  infection. A variety of host inflammatory mediators and substances such as, cytokines (TNF-α) and growth factors are released during infection and inflammation. These cytokines and growth factors affect the synthesis and secretion of pulmonary surfactant by pulmonary epithelial cells [ 32 , 33 ]. In the present study we found slightly increased levels of SP-D in  C. posadasii  protected mice, but not of SP-A (Fig  2 ). We speculate that the difference could be due to diverse mechanisms for regulation of SP-A and SP-D expression. Probably the cytokines and chemokines that are released as a result of FKS vaccination (protective immune response) increase the SP-D expression, but do not affect the SP-A expression. As reported earlier, the expression of SP-A and SP-D is differentially regulated during lung infection [ 34 ]. In future, more studies are warranted to understand the mechanism of the alterations in levels of surfactant phospholipids, SP-A and SP-D. At present, the treatment of  C. posadasii  infected patients with disseminated disease is not very effective [ 35 ]. Treatment with anti-fungal agents is most often related to relapse of the infection and side effects on the body. Earlier, the FKS based immunization has been found protective against  Coccidioides  infection in murine model of coccidioidomycosis, but failed in humans [ 35 ]. More pre-clinical studies on developing different vaccine strategies are underway. Since  C. posadasii  and  C. immitis  are highly virulent organisms, cause endemic infection, and pose a risk for bioterrorism, there is an urgent need for discovery of improved therapeutic drugs and regimens or preventive vaccines [ 3 , 35 ].
Discussion
In the present study we found that the levels of pulmonary surfactant collectins were altered in the lungs of  C. posadasii  infected mice, but were intact in lungs of  C. posadasii  protected mice after immunization with protective FKS vaccine. Furthermore, our results suggest that both SP-A and SP-D bind to Coccidioidal antigens. This is the first study where the amounts of SP-A and SP-D were measured in BALF samples from mice infected with lethal dose of  C. posadasii  and the binding of pulmonary collectins to Coccidioidal antigens was assessed. Since lung is the first organ of the body that comes into contact with air-borne  C. posadasii  arthroconidia, we hypothesized that the pulmonary surfactant may play an important role in regulating the immune response against  C. posadasii . Among four surfactant proteins, SP-A and SP-D, interact with most of the clinically important fungal pathogens including  Pneumocystis carinii  [ 23 ] Cryptococcus neoformans  [ 24 ],  Aspergillus fumigatus  [ 25 ] and  Candida albicans  [ 26 ]. SP-D has been shown to bind to  C. albicans  and directly inhibit growth by aggregation of the organism without involvement of macrophage dependent phagocytosis [ 27 ]. On the other hand, surfactant proteins also induce phagocytosis, activation and killing of  A. fumigatus  conidia and  C. neoformans  by alveolar macrophages and neutrophils [ 28 , 29 ]. In support of these findings, further evidence comes from a study by Madan et al., that suggests that the introduction of recombinant SP-D improves the lung function and increases the survival rate of mice infected with  A. fumigatus  [ 25 ]. The decrease in amounts of SP-A and SP-D during  C. posadasii  infection versus the unaltered amounts in the lungs of protected mice and binding of collectins to Coccidioidal antigens indicate that pulmonary collectins may be involved in uptake/phagocytosis of  C. posadasii  by antigen presenting cells and downstream immune regulation.C. posadasiiC. posadasiiC. posadasiiC. posadasiiC. posadasiiPneumocystis cariniiCryptococcus neoformansAspergillus fumigatusCandida albicansC. albicansA. fumigatusC. neoformansA. fumigatusC. posadasiiC. posadasiiBesides changes in amounts of SP-A and SP-D, a decrease in the amount of BALF phospholipids was also observed in  C. posadasii  infected mice (Table  2 ). Earlier, Sheehan et al., [ 29 ] and Hoffman et al., [ 30 ] have reported similar findings of reduced surfactant phospholipid level in BALF samples of rats and humans infected with  Pneumocystis carinii  [ 29 , 30 ]. To date, however, the information is lacking concerning how surfactant phospholipids may be involved in host defense [ 31 ]. Likewise, the mechanisms underlying the decrease of BALF surfactant in murine model of Coccidioidomycosis remain to be defined. We speculate that the reduction in the collectins and phospholipids could be either due to metabolic dysfunction of pulmonary type II epithelial cells during  C. posadasii  infection or due to their utilization in the binding and uptake of  C. posadasii  by local antigen presenting cells. The metabolic pathways of pulmonary type II epithelial cells may be affected by secondary inflammatory mediators, such as TNF-α or IL-1β, secreted by inflammatory cells during  C. posadasii  infection. A variety of host inflammatory mediators and substances such as, cytokines (TNF-α) and growth factors are released during infection and inflammation. These cytokines and growth factors affect the synthesis and secretion of pulmonary surfactant by pulmonary epithelial cells [ 32 , 33 ]. In the present study we found slightly increased levels of SP-D in  C. posadasii  protected mice, but not of SP-A (Fig  2 ). We speculate that the difference could be due to diverse mechanisms for regulation of SP-A and SP-D expression. Probably the cytokines and chemokines that are released as a result of FKS vaccination (protective immune response) increase the SP-D expression, but do not affect the SP-A expression. As reported earlier, the expression of SP-A and SP-D is differentially regulated during lung infection [ 34 ]. In future, more studies are warranted to understand the mechanism of the alterations in levels of surfactant phospholipids, SP-A and SP-D.C. posadasiiPneumocystis cariniiC. posadasiiC. posadasiiC. posadasiiC. posadasiiAt present, the treatment of  C. posadasii  infected patients with disseminated disease is not very effective [ 35 ]. Treatment with anti-fungal agents is most often related to relapse of the infection and side effects on the body. Earlier, the FKS based immunization has been found protective against  Coccidioides  infection in murine model of coccidioidomycosis, but failed in humans [ 35 ]. More pre-clinical studies on developing different vaccine strategies are underway. Since  C. posadasii  and  C. immitis  are highly virulent organisms, cause endemic infection, and pose a risk for bioterrorism, there is an urgent need for discovery of improved therapeutic drugs and regimens or preventive vaccines [ 3 , 35 ].C. posadasiiCoccidioidesC. posadasiiC. immitisConclusions In future, clearance experiments after  in vivo  administration of artificial or natural surfactant in  C. posadasii  infected mice may be useful in determining their therapeutic usefulness. We speculate that the findings from our present study would initiate similar studies to understand the role of pulmonary innate immune components in infectious diseases caused by  C. posadasii  or other virulent respiratory pathogens.
Conclusions
In future, clearance experiments after  in vivo  administration of artificial or natural surfactant in  C. posadasii  infected mice may be useful in determining their therapeutic usefulness. We speculate that the findings from our present study would initiate similar studies to understand the role of pulmonary innate immune components in infectious diseases caused by  C. posadasii  or other virulent respiratory pathogens.in vivoC. posadasiiC. posadasiiAuthors' Contributions SA designed and co-ordinated the study, performed assays and statistical analysis and drafted the manuscript. JJC assessed lung pathology. DMM prepared and immunized mice with FKS. All authors read and approved the final manuscript.
Authors' Contributions
SA designed and co-ordinated the study, performed assays and statistical analysis and drafted the manuscript. JJC assessed lung pathology. DMM prepared and immunized mice with FKS. All authors read and approved the final manuscript.


People are unpredictable. One night you may crave Italian food, but another only Thai will do. One day you might finish a crossword puzzle in record time, and the next not a single clue prompts an answer. Such behavioral variation has been found in laboratory studies, too: a person's ability to find a faint image on a screen varies widely from one viewing to the next. Similarly, when an animal repeatedly receives the same stimulus—for example, a faint image—a neuron in a region of the animal's visual brain might be very active upon one presentation and relatively quiet the next.Across the cerebral cortex—the brain region that integrates the senses and controls voluntary movement—neurons are notorious for their unpredictable behavior. The neurons themselves don't create this noise; when directly stimulated with an electrode multiple times, neurons will give the same response every time. Most neurons, however, receive signals from a host of other neurons. These various signals combine to form a seemingly noisy electrical input, which shows up as fluctuations in the recipient neuron's membrane potential—a difference in electrical charge between the inside and outside of the cell's membrane. Neuron function is intimately tied to the membrane potential, which is usually maintained within a narrow range, called the resting potential. But incoming signals can push the resting potential higher or lower. If the membrane potential rises above a certain threshold, the neuron fires, sending an electrical signal down its length. In this way, the brain relays and processes information.Since the 1960s, neuroscientists trying to account for the cortex's variable responses have pointed to noisy inputs from other parts of the brain as the prime suspect. In this issue of  PLoS Biology , Matteo Carandini addresses this longstanding mystery of neuron variability and comes up with a different answer. Carandini simultaneously measured the membrane potentials and firing patterns of individual neurons in the cat visual cortex. He found, surprisingly, that the membrane potentials varied much less than the firing patterns, ruling out noisy inputs as the cause of neurons' noisy outputs. Instead, the neurons amplified noise in the signals they received.PLoS BiologyCarandini then used a simple model of neuron behavior to explain why this would occur. He started with a tried-and-true approximation of neuron behavior, called the rectification model: a neuron doesn't fire until its membrane potential rises above a threshold, but once it crosses this threshold, its firing rate is correlated with the strength of incoming signals. Then he added the assumption that the neurons receive signals with some randomness. Given these minimal assumptions, Carandini showed that neurons fed a noisy signal will tend to amplify the noise in the signal. Importantly, his model reproduced a well-known phenomenon: as cortical neurons' average firing rate goes up, their firing rate also becomes more variable—that is, they get noisier.Carandini's model also predicted something new: as the firing rate continues to increase, the firing rate should become more consistent and less noisy—which he calls saturation of variability. Carandini's measurements in cats showed neurons actually behave this way, a key validation of his model.It's not clear whether this amplification of variability is something that helps or hampers the brain. Despite being a nuisance to neuroscientists, such fluctuations could be crucial to how the brain functions, Carandini speculates. Without some variability in their cortex, animals would act like cameras or other simple machines that respond the same way each time to a stimulus. It's advantageous for behavior, and hence brains, to be adaptable. But amplifying noise in a signal seems to run counter to relaying and processing the information in the signal. Carandini suggests that what appears as noise in the experiments are signals from other parts of the cortex—that is, noise is in the eye of the beholder. Now that the source of the variability is clear, neuroscientists can study whether it serves a function in the brain.


Lamptey and Wilson discuss the implications of a new study showing that combining treatment with prevention is the best approach to tackling the HIV pandemic.

There has been a recent and dramatic rise in global funding for HIV/AIDS, from US$2.1 billion in 2001 to US$6.1 billion in 2004 [ 1 ], thanks to several new funding mechanisms ( Box 1 ). These funds, coupled with reduced drug costs, make it feasible to roll out antiretroviral therapy (ART) even in resource-poor settings. Nevertheless, the total number of people living with HIV rose in 2004 to reach its highest level ever: an estimated 39.4 million people are living with the virus, including 4.9 million who acquired it in 2004 [ 1 ]. Therefore, the debate over the appropriate distribution of money between prevention efforts (such as voluntary counseling and testing [VCT], or behavior change) and treatment efforts (the provision of ART) is now more topical than ever.Box 1. Initiatives to Fund ART

 The WHO “3 by 5” Initiative:  This initiative aims to place 3 million people in low- and middle-income countries on ART by the end of 2005.
 The US President's Emergency Plan for HIV/AIDS Relief:  This initiative aims to treat 2 million HIV-infected people with ART, to prevent 7 million new infections, and to care for 10 million HIV-infected individuals and AIDS orphans in five years (2004–2009).
 The Global Fund to Fight AIDS, Tuberculosis, and Malaria:  In the five years following its inception (2002–2007), the Global Fund aims to provide 1.6 million people with ART and 52 million people with VCT, and to support more than 1 million orphans with medical services, education, and community care.
Box 1. Initiatives to Fund ART
The WHO “3 by 5” Initiative:  This initiative aims to place 3 million people in low- and middle-income countries on ART by the end of 2005.
 The US President's Emergency Plan for HIV/AIDS Relief:  This initiative aims to treat 2 million HIV-infected people with ART, to prevent 7 million new infections, and to care for 10 million HIV-infected individuals and AIDS orphans in five years (2004–2009).
 The Global Fund to Fight AIDS, Tuberculosis, and Malaria:  In the five years following its inception (2002–2007), the Global Fund aims to provide 1.6 million people with ART and 52 million people with VCT, and to support more than 1 million orphans with medical services, education, and community care.The WHO “3 by 5” Initiative:  This initiative aims to place 3 million people in low- and middle-income countries on ART by the end of 2005.The WHO “3 by 5” Initiative:The US President's Emergency Plan for HIV/AIDS Relief:  This initiative aims to treat 2 million HIV-infected people with ART, to prevent 7 million new infections, and to care for 10 million HIV-infected individuals and AIDS orphans in five years (2004–2009).The US President's Emergency Plan for HIV/AIDS Relief:The Global Fund to Fight AIDS, Tuberculosis, and Malaria:  In the five years following its inception (2002–2007), the Global Fund aims to provide 1.6 million people with ART and 52 million people with VCT, and to support more than 1 million orphans with medical services, education, and community care.The Global Fund to Fight AIDS, Tuberculosis, and Malaria:Balancing Prevention and Treatment The scale of the proposed increase in the number of patients receiving ART raises numerous questions about the treatment itself. Which drugs will be used? How much will it cost? How will their quality be monitored and assured? How will they be distributed? Who will be eligible? How will the desired level of treatment be sustained? Is there adequate infrastructure and human resources to support the expanded services? The commitment of substantial funding to treatment in resource-poor countries also has implications for the prevention efforts in those same countries. In many Western countries and Brazil (the sources of the majority of the available data on the subject), the impressive drop in mortality due to HIV following increased access to ART is coupled with a disheartening rise in the number of new cases of HIV, as emphasis and funding are shifted from prevention to treatment [ 2 ]. Countries in which this pattern has been seen are evidence of the pitfalls of failing to adapt prevention efforts once life-extending treatment becomes widely available. Of course, prevention and treatment are not mutually exclusive. Successful prevention efforts mean fewer patients will need the costly drug treatment programs, helping extend the sustainability of ART. In turn, the success of ART in prolonging healthy living helps prevention efforts by reducing the stigma associated with self-education and responsible behaviors.
Balancing Prevention and Treatment
The scale of the proposed increase in the number of patients receiving ART raises numerous questions about the treatment itself. Which drugs will be used? How much will it cost? How will their quality be monitored and assured? How will they be distributed? Who will be eligible? How will the desired level of treatment be sustained? Is there adequate infrastructure and human resources to support the expanded services?The commitment of substantial funding to treatment in resource-poor countries also has implications for the prevention efforts in those same countries. In many Western countries and Brazil (the sources of the majority of the available data on the subject), the impressive drop in mortality due to HIV following increased access to ART is coupled with a disheartening rise in the number of new cases of HIV, as emphasis and funding are shifted from prevention to treatment [ 2 ]. Countries in which this pattern has been seen are evidence of the pitfalls of failing to adapt prevention efforts once life-extending treatment becomes widely available.Of course, prevention and treatment are not mutually exclusive. Successful prevention efforts mean fewer patients will need the costly drug treatment programs, helping extend the sustainability of ART. In turn, the success of ART in prolonging healthy living helps prevention efforts by reducing the stigma associated with self-education and responsible behaviors.Measuring Prevention and Treatment Effects In their study in the January 2005 issue of  PLoS Medicine , “Integrating HIV Prevention and Treatment: From Slogans to Impact,” Salomon and colleagues use mathematical modeling to assess the epidemiologic impact of treatment and prevention efforts, and to quantify the opportunities and potential risks of large-scale treatment roll-out. Using a variety of different scenarios, they propose methods for establishing the most effective balance between spending on prevention and spending on treatment. Modeling is a technique used by many scientists, including epidemiologists and statisticians, to create a mathematical equation that can be used to determine which variables affect an outcome of interest, and to what extent. Once the influential variables are determined, a baseline model is established that includes those variables and reflects their relative importance to the outcome. The effect of changing the value of any of these variables, or several of them, can then be tested, and new outcomes projected. HIV modeling is inexact and requires far better data but can nevertheless provide important insights. Salomon and colleagues used mathematical modeling to assess the effect of changing aspects of the HIV/AIDS “equation” on the future course of the HIV/AIDS epidemic. First, a baseline model was created to fit expected HIV/AIDS projections for the year 2020 if there were to be no change in the current epidemiologic trends—no ART scale-up, and no changes in prevention efforts or behavior. Heterosexual contact is the predominant mode of HIV transmission across Africa, and Salomon and colleagues' study modeled the disease only within the heterosexual population. The model was also tailored to take into account epidemiologic, demographic, and sociologic patterns in the eastern, central/western and southern regions of Africa. Using the baseline models tailored to each region, the effects of prevention and treatment efforts were then measured. Two treatment-centered scenarios were tested in which the World Health Organization's “3 by 5” initiative (see  Box 1 ) was achieved. In these treatment-centered scenarios, the reduction of transmissibility, the number of partners of each patient, and condom use were either optimal (reduced transmissibility, reduced partners, and increased condom use) or less than optimal. The prevention-centered scenario tested the impact of a comprehensive package of 12 prevention tools (such as VCT and peer counseling for sex workers), modeling only partial effectiveness at the population level, to reflect weaker political and social support for HIV control efforts. Finally, combined response scenarios were tested. In the first scenario, treatment efforts strengthened prevention efforts as, for example, when the availability of ART increases people's willingness to undergo testing. In the second, an emphasis on treatment led to less effective implementation of prevention efforts. Baseline projections in Salomon and colleagues' study showed that without any behavioral change or ART scale-up, the HIV/AIDS prevalence rate would remain relatively stable, but the number of new infections would increase by 52.3 million by 2020. Treatment-centered scenarios reduced the total number of new infections through 2020 by a maximum of 3 million, or 6%, while indicating that the number of AIDS deaths through 2020 would decline by 13%, to 32.4 million. A prevention-centered strategy would provide greater reductions in incidence (36%) and similar mortality reductions by 2020, but more modest mortality benefits over the next five to ten years. The scenarios in which all of these statistics were most improved, however, were those that combined both prevention and treatment efforts. In the scenario in which treatment enhanced prevention, Salomon and colleagues projected 29 million averted infections (55%) and 10 million averted deaths (27%) through the year 2020. However, if a narrow focus on treatment scale-up leads to reduced effectiveness of prevention efforts, the benefits of a combined response would be considerably smaller—9 million averted infections (17%) and 6 million averted deaths (16%) ( Figure 1 ). Figure 1 Projected New Adult Infections and Total Adult Deaths, in Millions, to 2020 This graph represents projections through 2020, and, when there was a choice, highlights the more successful iteration of a model. The treatment-centered response, therefore, shows data from the optimal-effects model, and the combined response data reflect the optimistic model. Combining treatment with effective prevention efforts could reduce the resource needs for treatment dramatically in the long term. In the various scenarios the numbers of people being treated in 2020 ranges from 9.2 million in a treatment-only scenario with mixed effects, to 4.2 million in a combined response with positive treatment–prevention synergies.
Measuring Prevention and Treatment Effects
In their study in the January 2005 issue of  PLoS Medicine , “Integrating HIV Prevention and Treatment: From Slogans to Impact,” Salomon and colleagues use mathematical modeling to assess the epidemiologic impact of treatment and prevention efforts, and to quantify the opportunities and potential risks of large-scale treatment roll-out. Using a variety of different scenarios, they propose methods for establishing the most effective balance between spending on prevention and spending on treatment.PLoS MedicineModeling is a technique used by many scientists, including epidemiologists and statisticians, to create a mathematical equation that can be used to determine which variables affect an outcome of interest, and to what extent. Once the influential variables are determined, a baseline model is established that includes those variables and reflects their relative importance to the outcome. The effect of changing the value of any of these variables, or several of them, can then be tested, and new outcomes projected. HIV modeling is inexact and requires far better data but can nevertheless provide important insights.Salomon and colleagues used mathematical modeling to assess the effect of changing aspects of the HIV/AIDS “equation” on the future course of the HIV/AIDS epidemic. First, a baseline model was created to fit expected HIV/AIDS projections for the year 2020 if there were to be no change in the current epidemiologic trends—no ART scale-up, and no changes in prevention efforts or behavior. Heterosexual contact is the predominant mode of HIV transmission across Africa, and Salomon and colleagues' study modeled the disease only within the heterosexual population. The model was also tailored to take into account epidemiologic, demographic, and sociologic patterns in the eastern, central/western and southern regions of Africa. Using the baseline models tailored to each region, the effects of prevention and treatment efforts were then measured.Two treatment-centered scenarios were tested in which the World Health Organization's “3 by 5” initiative (see  Box 1 ) was achieved. In these treatment-centered scenarios, the reduction of transmissibility, the number of partners of each patient, and condom use were either optimal (reduced transmissibility, reduced partners, and increased condom use) or less than optimal. The prevention-centered scenario tested the impact of a comprehensive package of 12 prevention tools (such as VCT and peer counseling for sex workers), modeling only partial effectiveness at the population level, to reflect weaker political and social support for HIV control efforts. Finally, combined response scenarios were tested. In the first scenario, treatment efforts strengthened prevention efforts as, for example, when the availability of ART increases people's willingness to undergo testing. In the second, an emphasis on treatment led to less effective implementation of prevention efforts.Baseline projections in Salomon and colleagues' study showed that without any behavioral change or ART scale-up, the HIV/AIDS prevalence rate would remain relatively stable, but the number of new infections would increase by 52.3 million by 2020. Treatment-centered scenarios reduced the total number of new infections through 2020 by a maximum of 3 million, or 6%, while indicating that the number of AIDS deaths through 2020 would decline by 13%, to 32.4 million. A prevention-centered strategy would provide greater reductions in incidence (36%) and similar mortality reductions by 2020, but more modest mortality benefits over the next five to ten years.The scenarios in which all of these statistics were most improved, however, were those that combined both prevention and treatment efforts. In the scenario in which treatment enhanced prevention, Salomon and colleagues projected 29 million averted infections (55%) and 10 million averted deaths (27%) through the year 2020. However, if a narrow focus on treatment scale-up leads to reduced effectiveness of prevention efforts, the benefits of a combined response would be considerably smaller—9 million averted infections (17%) and 6 million averted deaths (16%) ( Figure 1 ).Combining treatment with effective prevention efforts could reduce the resource needs for treatment dramatically in the long term. In the various scenarios the numbers of people being treated in 2020 ranges from 9.2 million in a treatment-only scenario with mixed effects, to 4.2 million in a combined response with positive treatment–prevention synergies.Moving Forward The authors have demonstrated through mathematical modeling that the integration of treatment and prevention is epidemiologically sound. However, an integrated and comprehensive program ( Figure 2 ) is not only logical but makes sense from the service delivery point of view: it can be cost-effective and ideal for the community. Figure 2 Components of an Integrated Comprehensive HIV/AIDS Program PMTCT, prevention of mother-to-child transmission; STD, sexually transmitted disease.
 Effective prevention makes treatment more affordable and sustainable.  Effective prevention can lead to a substantial reduction in the number of new infections and therefore ultimately will lead to a reduction in the number of people who will need treatment. The reduction of adult HIV/AIDS prevalence in Uganda from 18.5% to 6% over the last several years has reduced the number of those eventually needing treatment by nearly 68% [ 3 ]. Unless the incidence of HIV is sharply reduced, HIV treatment will not be able to keep pace with all those who will need therapy [ 4 ]. Salomon and colleagues' reaffirmation that only effective prevention will make treatment affordable is critically important.
 Successful treatment and care can make prevention more acceptable and effective.  Widespread access to treatment could bring millions of people into health-care settings, providing new opportunities for health-care workers to deliver and reinforce HIV prevention messages and interventions [ 4 ]. Improved access to HIV testing provides an entry point to both prevention and treatment services and provides a unique opportunity to identify and target the infected, vulnerable, and uninfected with more appropriate interventions. All health-care settings, including HIV treatment sites, should deliver HIV prevention services [ 4 ].
 Prevention can make treatment more accessible.  The early establishment of community-based prevention services in rural Ghana was instrumental in reducing the stigma of AIDS and improving the knowledge and attitude of the community prior to the development of ART and VCT services (K. Torpey, personal communication). This process also made it easier for community and implementing agencies to identify and refer patients needing treatment services.
 Expanded care and prevention activities have synergistic effects.  Continued effective treatment, care, and prevention programs will reduce the number of orphans and vulnerable children, reduce mother to child transmission of the virus, and improve the lives of families and the strength of communities.
 Integration ensures that prevention activities are not neglected.  The world has a unique opportunity, as ART services are launched and expanded, to simultaneously bolster prevention efforts [ 4 ]. Experience in the United States indicates that availability of treatment can lead to increased risk behavior [ 5 ]. In addition, the improvement in the health, well-being, and longevity of people living with AIDS could increase the opportunities for HIV transmission. Integration can help reduce these potential negative impacts of treatment.
 Integration can provide opportunities to address vulnerable groups more effectively.  A commitment to providing large-scale treatment helps to focus attention on communities at greatest risk, particularly in lower prevalence contexts. This provides an opportunity to address the prevention and treatment needs of vulnerable groups more effectively.
 Treatment resources can help improve infrastructure for prevention and other health services.  The training of health providers and improvements in laboratory services, pharmacy, logistics, commodity management, and health information systems can benefit both treatment and prevention services. Further, in many countries, a large number of health-care workers are themselves infected. Treatment can help to preserve the lives and productivity of these critically needed AIDS prevention and treatment workers, as well as those of other health professionals.
 A long-term decline in AIDS deaths may be preventing new infections.  The short-term decline in AIDS deaths is driven by effective care and treatment programs, but a long-term decline may be driven by the prevention of new infections. Integrated and comprehensive strategies are more likely to lead to affordable, sustainable programs.
 Success requires dramatic expansion of both ART and prevention.  Globally, fewer than one in five people at high risk of infection have access to proven HIV prevention interventions [ 6 ] and less than 10% have access to ART [ 1 ]. Unless there is a substantial increase in commitment and resources for both prevention and ART, efforts to control HIV/AIDS and mitigate its impact will only meet with partial and limited success. In addition, to increase resources, intensified commitment is required to ensure every opportunity is taken to integrate prevention and treatment. Future analysis and debate should move from comparisons of prevention and treatment priorities to a sustained analysis of how we can reciprocally integrate and strengthen prevention and care and use every opportunity provided by one to reinforce the other. We must focus on the development of training, monitoring, and quality assurance systems that ensure that prevention and care are integrated whenever possible.
 The results of Salomon and colleagues' model need to be validated.  Further operational research is needed to validate the findings of this study.
Moving Forward
The authors have demonstrated through mathematical modeling that the integration of treatment and prevention is epidemiologically sound. However, an integrated and comprehensive program ( Figure 2 ) is not only logical but makes sense from the service delivery point of view: it can be cost-effective and ideal for the community.Effective prevention makes treatment more affordable and sustainable.  Effective prevention can lead to a substantial reduction in the number of new infections and therefore ultimately will lead to a reduction in the number of people who will need treatment. The reduction of adult HIV/AIDS prevalence in Uganda from 18.5% to 6% over the last several years has reduced the number of those eventually needing treatment by nearly 68% [ 3 ]. Unless the incidence of HIV is sharply reduced, HIV treatment will not be able to keep pace with all those who will need therapy [ 4 ]. Salomon and colleagues' reaffirmation that only effective prevention will make treatment affordable is critically important.Effective prevention makes treatment more affordable and sustainable.Successful treatment and care can make prevention more acceptable and effective.  Widespread access to treatment could bring millions of people into health-care settings, providing new opportunities for health-care workers to deliver and reinforce HIV prevention messages and interventions [ 4 ]. Improved access to HIV testing provides an entry point to both prevention and treatment services and provides a unique opportunity to identify and target the infected, vulnerable, and uninfected with more appropriate interventions. All health-care settings, including HIV treatment sites, should deliver HIV prevention services [ 4 ].Successful treatment and care can make prevention more acceptable and effective.Prevention can make treatment more accessible.  The early establishment of community-based prevention services in rural Ghana was instrumental in reducing the stigma of AIDS and improving the knowledge and attitude of the community prior to the development of ART and VCT services (K. Torpey, personal communication). This process also made it easier for community and implementing agencies to identify and refer patients needing treatment services.Prevention can make treatment more accessible.Expanded care and prevention activities have synergistic effects.  Continued effective treatment, care, and prevention programs will reduce the number of orphans and vulnerable children, reduce mother to child transmission of the virus, and improve the lives of families and the strength of communities.Expanded care and prevention activities have synergistic effects.Integration ensures that prevention activities are not neglected.  The world has a unique opportunity, as ART services are launched and expanded, to simultaneously bolster prevention efforts [ 4 ]. Experience in the United States indicates that availability of treatment can lead to increased risk behavior [ 5 ]. In addition, the improvement in the health, well-being, and longevity of people living with AIDS could increase the opportunities for HIV transmission. Integration can help reduce these potential negative impacts of treatment.Integration ensures that prevention activities are not neglected.Integration can provide opportunities to address vulnerable groups more effectively.  A commitment to providing large-scale treatment helps to focus attention on communities at greatest risk, particularly in lower prevalence contexts. This provides an opportunity to address the prevention and treatment needs of vulnerable groups more effectively.Integration can provide opportunities to address vulnerable groups more effectively.Treatment resources can help improve infrastructure for prevention and other health services.  The training of health providers and improvements in laboratory services, pharmacy, logistics, commodity management, and health information systems can benefit both treatment and prevention services. Further, in many countries, a large number of health-care workers are themselves infected. Treatment can help to preserve the lives and productivity of these critically needed AIDS prevention and treatment workers, as well as those of other health professionals.Treatment resources can help improve infrastructure for prevention and other health services.A long-term decline in AIDS deaths may be preventing new infections.  The short-term decline in AIDS deaths is driven by effective care and treatment programs, but a long-term decline may be driven by the prevention of new infections. Integrated and comprehensive strategies are more likely to lead to affordable, sustainable programs.A long-term decline in AIDS deaths may be preventing new infections.Success requires dramatic expansion of both ART and prevention.  Globally, fewer than one in five people at high risk of infection have access to proven HIV prevention interventions [ 6 ] and less than 10% have access to ART [ 1 ]. Unless there is a substantial increase in commitment and resources for both prevention and ART, efforts to control HIV/AIDS and mitigate its impact will only meet with partial and limited success. In addition, to increase resources, intensified commitment is required to ensure every opportunity is taken to integrate prevention and treatment. Future analysis and debate should move from comparisons of prevention and treatment priorities to a sustained analysis of how we can reciprocally integrate and strengthen prevention and care and use every opportunity provided by one to reinforce the other. We must focus on the development of training, monitoring, and quality assurance systems that ensure that prevention and care are integrated whenever possible.Success requires dramatic expansion of both ART and prevention.The results of Salomon and colleagues' model need to be validated.  Further operational research is needed to validate the findings of this study.The results of Salomon and colleagues' model need to be validated.