The pre- and post-chemotherapy design is based upon known in vivo kinetics and immune modulatory effects of these molecules. Carboplatin (Paraplatin®) was selected as the cornerstone of treatment of epithelial ovarian cancer (EOC).Monocyte/macrophages (MO/MA), a polymorphic population of innate immune cells, have the potential to mediate antitumor effects, and may also contribute to protumor effects. A priming and post-chemotherapy schedule of the myeloid cell mobilizing and immune stimulatory growth factor, granulocyte monocyte stimulating factor  for 7 days plus subcutaneous rIFN-γ1b (100 μg) on days 5 and 7, before and after intravenous carboplatin (area under the curve of 5). We performed standard hematologic assessment and monitored monocyte (MO), dendritic cell, major cell subset counts, and antibody-dependent cell-mediated cytotoxicity (ADCC) against a Her2neuP ≤ .001 for each); the proportion of platelets increased 9 days after the second (P ≤ .002) and third (P ≤ .04) carboplatin treatments; and the number of cells in the activated MO subsets CD14+HLA-DR+, CD14+CD64+, and CD14+CXCR3+ increased (P ≤ .04 for each); plasma levels of the proangiogenic interleukins 1α, 6, and 8 were lower (P ≤ .03 for each); M-CSF, a product of activated MO/MA, was increased on day 9 (P = .007); and GM-CSF was increased in plasma after GM-CSF administration (P ≤ .04). Quality of life measurements were reduced during the GM-CSF/IFN-γ1b cycle while recovering at pre-chemotherapy baseline for FACT-G scores only.Our analysis comprised only the first 3 months of treatment in the initial 25 patients. Relative to pretreatment baseline values, white blood cell, neutrophil, MO, and eosinophil counts increased (A novel regimen of GM-CSF plus IFN-γ1b administered to 25 EOC patients receiving carboplatin increased myeloid cells, platelets and total activated MO populations during the initial 3 months; however, ADCC responses were not consistently enhanced during this period. In 2005 there will be an estimated 22,200 new cases of epithelial ovarian cancer (EOC) and 16,210 deaths from it in the United States, where EOC is the fifth most common cause of cancer-related death in women ,4. RecurMonocytes and macrophages (MO/MAs) are the most common innate cell population in humans. Their role in cancer patients is complex because they may either enhance or impair immunity. Granulocyte-monocyte colony-stimulating factor (GM-CSF) and recombinant interferon gamma 1b (IFN-γ1b) are commercially available cytokines that can modulate MO/MA activity. GM-CSF can mobilize and mature myeloid cells, including MO/MAs and dendritic cells (DCs) ,9, whereAt biologically active doses, GM-CSF increases both neutrophil and MO counts to reach a plateau within 5 to 10 days . When adin vitro by stimulated peripheral blood MOs , and IFN-γ1b) and provided some treatment effects against metastatic renal tumors and melanoma [GM-CSF may also stimulate the immune system by enhancing antitumor effects by either innate or adaptive antitumor immunity ,16. Thuslood MOs , enhancelood MOs ,19, and lood MOs , GM-CSF lood MOs  and in blood MOs . GM-CSF melanoma ,23. OptiWe designed a clinical trial of two immunomodulatory cytokines, GM-CSF and IFN-γ1b, administered both before and after carboplatin treatment in patients with platinum-sensitive EOC that had recurred. We expected that IFN-γ1b  would enhance the activation of MO/MAs while GM-CSF would exert its proliferative effects on myeloid progenitor cells, minimizing any possible antiproliferative effects from the IFN-γ1b. Here we report the hematopoietic and myeloid immune cell effects during the first three courses of a chemoimmunotherapy regimen that consisted of GM-CSF and IFN-γ1b priming, carboplatin, and post-carboplatin GM-CSF and IFN-γ1b. Detailed analysis of the response and toxicity of the ongoing phase II trial will be reported after the trial's completion.Patients eligible for inclusion in the study had Müllerian carcinoma  that had initially responded to platinum-based chemotherapy but recurred after a treatment-free interval of ≥ 6 months; these tumors were considered "potentially platinum sensitive." Patients were also required to have radiographically or clinically measurable disease and CA-125 determinations. Adequate hematologic, renal, and hepatic function was required as defined by: absolute neutrophil count ≥ 1,500 cells/μL, platelet count ≥ 100,000 cells/μL, serum creatinine ≤ 1.5 mg/dL, serum bilirubin ≤ 1.5 mg/dL, and aspartate transaminase ≤ 2.5 × normal. A Zubrod performance status score of ≤ 2 was also required. Patients could not have received more than two prior chemotherapy regimens (first-line platinum and platinum re-induction were counted as one regimen). Other exclusion criteria included prior immunotherapy, abdominal radiotherapy, active heart or autoimmune disease, inflammatory bowel disease, brain metastases, serum albumin ≤ 3 g/dL, or known hypersensitivity to platinum agents. All patients provided informed consent for participation in this IRB approved study.®, specific activity 5.6 × 106 IU/mg; Berlex Labs, Seattle, WA) was provided in vials containing either 250 or 500 μg of the lyophilized powder and 40 mg of mannitol, 10 mg of sucrose, and 1.2 mg of tromethamine. The cytokine was reconstituted with 1.0 mL of either bacteriostatic water or sterile water at room temperature.GM-CSF  was provided in 0.5-mL vials containing 100 μg of IFN-γ1b, 20 mg of mannitol, 0.36 mg of sodium succinate, and 0.05 mg of polysorbate-20. The cytokine was reconstituted in 0.5 mL of sterile water at room temperature.IFN-γ1b  at an area under the curve of 5 . Carboplatin was administered 48–60 hours after the last dose of GM-CSF. For the post-chemotherapy cycle, GM-CSF was started within 24–36 hours. For the first and second cycles of GM-CSF, the dose started at 400 μg per day and escalated to 600 μg per day if the MO count did not at least double and reach a level of 1,000 cells/μL. The GM-CSF dose could be reduced by 200 μg in response to grade 3 major organ toxicity. IFN-γ1b was administered by subcutaneous injection at a fixed dose of 100 μg on the fifth and seventh days of each 7-day cycle of GM-CSF. No dose escalation of IFN-γ1b was permitted.For each course of treatment, GM-CSF was administered by subcutaneous injection in two 7-day cycles, one preceding and the other one following a fixed intravenous dose of carboplatin  . Up to ti.e,. within 10 days before the first GM-CSF dose), the following were checked for each patient: complete blood count, serum creatinine, blood urea nitrogen, total bilirubin, aspartate transaminase, alanine transaminase, electrolytes , carbon dioxide, serum albumin, total protein, glucose, alkaline phosphate, lactate dehydrogenase, and CA-125. A complete blood count was also obtained on days -4, 1, 6, 9, 16, 21, 25, and 28 of the first treatment course to monitor the pre and post-chemotherapy GM-CSF/IFN-γ and on days 1, 9, 16, 21, and 28 of the second and third treatment courses.Before the start of each treatment course  was collected in ACD (yellow top) vacutainer tubes The blood was centrifuged, the plasma collected, and the mononuclear cells isolated by Ficoll-Hypaque density gradient separation. In addition, for cytotoxicity studies, MOs were obtained from anonymized normal donors through the blood bank as controls.In the 12 patients who consented to optional studies, peripheral blood was examined for the number and activation (or maturation) status of MOs and DCS and for bright leukocytes or CD14dim, CD45+ leukocytes. Mononuclear leukocytes were further characterized for coexpression of HLA-DR, CD64, and in some instances CD32 and CD32B. DCs were identified directly in mononuclear cell preparations of blood as lineage-negative, HLA-DR+ leukocytes by using multidimensional flow cytometry [+ pre-DC1 cells and CD123+ and pre-DC2 cell subsets [+ DCs were also tested for coexpression of this marker. CD16+ and CD16- MOs have been shown to give rise to DCs with different phenotypic and functional properties [MOs were identified within mononuclear cell preparations by flow cytometry as CD14ytometry ,26. Addi subsets . Becauseoperties . Therefooperties . For fun3H-TdR release assay we developed [3H-labeled thymidine  in 10 mL of RPMI 1640 medium with heat-inactivated 15% fetal bovine serum for 18 to 24 hours at 37°C in a humidified 5% CO2 incubator. Target cells were incubated with the humanized IgG1 antibody trastuzumab , which recognizes the Her2neu surface antigen, at a concentration of 5 μg/mL for 30 min at 37°C in a shaking water bath. Sensitized target cells were washed once and seeded with MO/MAs at an effects-to-target ratio of 20:1 in 96-well U-bottom microtiter plates. Purified MO were isolated from PBMC by negative selection, using a MO Isolation Kit and a MACS separator  as per manufacturer's instruction. MOs obtained from non-MCSF stimulated buffy coat samples were utilized as controls as previously described [6 cells for 1 hour. Plates were centrifuged at 1,000 rpm for 1 min and incubated for 60 to 72 hours at 37°C in a humidified 5% CO2 incubator.Enriched MOs from EOC patients undergoing treatment were tested for their ability to induce the cytotoxicity of tumor cells directly or in the presence of an antibody capable of mediating ADCC by using a eveloped ,31. Brieescribed  were inc3H-TdR release from target cells was counted in a WinSpectral liquid scintillation counter  for 1 min. The percent ADCC for MO/MAs was calculated as  ÷ (maximum release - spontaneous release) × 100, in which release values are in counts per minutes and experimental release is counts from cell-free culture supernatant from MO/MAs incubated with labeled tumor cells, spontaneous release is counts from cell-free culture supernatant from labeled tumor cells only, and maximum release is counts from cell-free culture supernatant from lysed (5% Triton) labeled tumor cells only.Plates were spun down at 1,000 rpm for 5 min at 25°C, and cell-free supernatants were transferred to scintillation vials into which 5 mL of scintillation fluid was added . Patients who consented to optional studies provided samples for measurement of plasma levels of cytokines IL-1α, IL-1β, IL-6, IL-10, and TNF-α and the chemokine CXCL8 (IL-8). Plasma samples were taken on days -8, -4, 1, and 9 of the first course and at 3 months after the first treatment course was begun. We used the Multiplex Bead chemoluminescence assay . The Multiplex Bead Immunoassay is a multiplex assay that uses the Luminex 100 analyzer  and associated software. Levels of multiple cytokines are measured in small volumes by using fluorescently encoded microspheres labeled with a distinguishable fluorophore that allows the microspheres to be gated to a particular region by the scanner. Antibodies specific for the cytokine or chemokine of interest  are covalently linked to individual beads of a different fluorescent marker, thereby causing the cytokines or chemokine to bind to the specific complementary antibody-coated bead and produce a unique fluorescent signature. The minimum detectable levels using this method are: IL1α, 5 pg/mL; IL-1β, 15 pg/mL; IL-6, 3 pg/mL; IL-10, 5 pg/mL; TNF-α, 10 pg/mL; CXCL8, 3 pg/mL; GM-CSF, 3 pg/mL; and M-CSF, 9 pg/mL.We used the Functional Assessment of Chronic Illness Therapy-Biologic Response Modifier (FACT-BRM version 4) to assess changes in patients' quality of life. This questionnaire is a 44-item, cancer-specific measure of health-related quality of life It consiWilcoxon rank sum tests were used to evaluate differences in variables between individual time points. No correction was made for multiple testing. Statistical significance was declared for p-values < 5%. SAS 9.1 was used for statistical analyses.Characteristics of the first 25 enrolled patients and their tumors and prior treatment regimens are summarized in Table P = .002) and third (P ≤ .04) treatment courses   count increased as expected after each GM-CSF cycle and peaked 6 days after the first dose of carboplatin Figure . Prechems Figure . When wens) Fig. .nd and 1 after 3rd course).Sixteen of the 25 (64%) patients required a dose escalation of GM-CSF from 400 to 600 μg. Twenty of the 25 (80%) patients met the target MO expansion of a doubling from day -8 and >1,000 cells/μL. For two patients, the dose was reduced from 600 to 400 μg because of toxicity, one before and one after the second course. In five of the 25 patients (20%), the presence of neutropenia or thrombocytopenia required that the day 7 dose of IFN-γ1b not be given , cells with high-affinity Fc receptor (FcR), i.e,. CD64+ cells (P = .01), and cells with chemokine receptor, i.e,. CXCR3+ cells (P ≤ .02). There were no significant differences in the absolute number of cells with low-affinity FcR , the absolute number of DCs , or the proportion of DC subsets. In addition, there were no significant alterations in the numbers or proportions of natural killer  cells over the time period under study (data not shown). The proportions of CD83+ DCs and CD2+ cells at day -8 were 1.4% and 3.7%, respectively.For 12 of the 25 patients (48%), MO and DC subsets from the first treatment course were examined as a function of time Figure . CompareWe also examined the CD32 MO population in a small sample of four patients. On day -8, the mean (± SD) number of CD32+ MO/MAs was 99.4 ± 0.4, and the mean fluorescent intensity (MFI) was 145 ± 51. On day 9, the mean number of CD32+ MO/MAs remained stable (99.9 ± 0.2) but the mean MFI increased to 250 ± 45.CD32B expression was also measured in a small sample of two patients. The mean expression level was 36 ± 5% on day -8 and 37.4 ± 0.2% on day 9, and the corresponding mean MFI values were 16 ± 1% and 22 ± 1.4%.in vitro MO-mediated tumor cell cytotoxicity and ADCC with purified MOs from EOC patients than with purified MOs from buffy coat samples of normal subjects . GM-CSF levels were increased at each post treatment time point, suggesting that daily doses of GM-CSF were associated with its detection in the circulation.Plasma levels of proinflammatory cytokines and the chemokine CXCL8 are shown in Table All 25 patients were evaluated for up to 3 months for toxicity. Treatment with the cytokines GM-CSF and IFN-γ1b was well tolerated overall. Consistent with prior reports, 18 patients (72%) initially experienced grade 3 fatigue. The fatigue did not recur in subsequent treatment courses in any patient. One patient withdrew after the second treatment course because of grade 3 fatigue and chest tightness attributed to GM-CSF treatment. One patient who developed grade 1 liver toxicity after the third treatment course had to be removed from study after the fifth treatment course. A liver biopsy examination suggested drug-induced injury, but a viral etiology could not be excluded since there was serologic evidence of prior cytomegalovirus exposure. Six of the 25 patients required carboplatin desensitization for an allergic reaction in the second or third treatment course. Two patients experienced a severe allergic reaction to carboplatin and were removed from the study after the third and fourth treatment course, respectively.v 81; P = .009) and BRM  scores compared with day -8. The day before the start of the second treatment course, the FACT-G scores returned to pretreatment baseline values, but the BRM scores remained significantly suppressed . A similar pattern was seen for the third treatment course. Symptoms specifically related to BRM never returned to pretreatment baseline levels.In the first treatment course, there was no significant change in FACT-G and BRM scores during cytokine administration before the infusion of carboplatin . On day 9, there was a statistically and clinically significant decrease in the FACT-G (90 Ten of the 25 patients (40%) had a clinical response, as based on measurable disease, after completion of three chemoimmunotherapy courses. Eighteen of these 25 patients (72%) had at least a 50% decline or normalization of the CA-125 level from pretreatment baseline through the end of the third treatment course, confirmed by two post-treatment readings.As stated earlier, the purpose of this paper is to describe the main hematologic and immunologic effects observed in the initial 25 patients of the ongoing phase II chemotherapy clinical trial. Since the trial is ongoing, reporting of response and toxicity information should be considered preliminary. To facilitate the statistical analysis, we selected only the first 3 courses of treatment.We have shown that 3 months of a prechemotherapy priming schedule of daily subcutaneous GM-CSF for 7 days followed by a postchemotherapy schedule of subcutaneous GM-CSF for 7 days was reasonably well tolerated. Increased absolute numbers of total WBCs, neutrophils and MOs were observed. GM-CSF is the only approved drug in the United States that can also stimulate non-granulocyte myeloid lineages such as MO/MAs and DCs.The platelet count after three chemoimmunotherapy courses was not significantly altered from the pretreatment baseline count, but we thought it remarkable that there were statistically significant increases in platelet levels on day 9 of the second and third treatment courses. To our knowledge, ours is the first report of a significantly higher platelet count after GM-CSF treatment than before GM-CSF treatment. This increase occurred at the same time as the counts of the myeloid lineages were increased and the levels of IL-6, which has platelet-stimulating properties, were decreased. An early study had reported higher platelet levels in a group of patients treated with postchemotherapy GM-CSF than in another patient group not treated with GM-CSF after chemotherapy The freqIn our study, the eosinophil count was increased on day 9 of all three treatment courses, but the count had returned to pretreatment baseline levels 3 months after the chemoimmunotherapy regimen had been started. An earlier study showed no association between the occurrence of hypersensitivity reactions to carboplatin and eosinophilia.One of the aims of our study was to determine whether priming and postchemotherapy treatment with cytokines GM-CSF and IFN-γ1b increases MO counts. We found that MO counts increased significantly on day 9 of each of the three treatment courses and that MO values almost doubled from day -8 to day 9 of each treatment course. Eighty percent of the patients met the criteria of a doubling of the MO count and >1,000 MOs/μL. From our results, daily subcutaneous administration of 400 μg of GM-CSF appears to be a reasonable starting dose and schedule in this previously treated patient population. In another study, although daily and twice daily GM-CSF dosing over 10 to 14 days was administered after carboplatin and cyclophosphamide treatment, the addition of a twice daily prechemotherapy priming schedule for 4 days resulted in unacceptable toxicity in a small cohort of patients.[in vitro study has suggested that IFN-γ plus IL-4 can induce maturation of myeloid DCs to a predominantly CD11C+ phenotype after GM-CSF administration[2O2 production and Fc-γ R expression by MOs, whereas clinical toxicity was low[2O2,[IFN-γ is the most potent clinically available activator of MO/MAs. In adaptive immunity, IFN-γ participates in the conversion of precytotoxic to cytotoxic effector T cells. IFN-γ also enhances natural cell activity and can inhibit tumor cell proliferation directly by a variety of mechanisms An in viistration We did ny was low In other low[2O2, although low[2O2, We consi low[2O2, and in p low[2O2,,48 A rec low[2O2, Because i.e,. cells with high-affinity FcRs) also increased, but there were no significant changes in CD16+ cells . In contrast, the mean fluorescent intensity (MFI) of CD32+ cells, which made up 100% of MOs before treatment with GM-CSF, increased significantly. Two previous studies had suggested that GM-CSF can stimulate antitumor functions, such as ADCC, or MO-mediated cytotoxicity[GM-CSF plus rIFN-γ1b also produced alterations in the activation cell surface marker phenotype of the MO population. Compared with pretreatment baseline, the proportion and number of HLA-DR+ cells increased significantly after GM-CSF treatment alone (day -4) or with IFN-γ1b (day 9) Fig. . The numotoxicity,50 We footoxicity We have Previous work has demonstrated that systemic administration of GM-CSF increases numbers of intraperitoneal MAs, which could be important in ovarian cancer because EOC is primarily a tumor of the peritoneal cavity In our sin vivo. In contrast, the plasma levels of CXCL8 and IL1α were significantly decreased at 3 months as a number of patients  were beginning to show responses.Measurements of cytokines can provide some indication of the patient's tumor environment and might also be useful for monitoring immunomodulatory alterations of the treatment. We showed here that there were no significant increases in the plasma levels of the proinflammatory cytokines IL-1α, IL-6, CXCL-8, or of TNF-α, even though these proteins are produced by activated MO/MA. This suggests that the combination of GM-CSF and IFN-γ1b may not directly enhance production of these proangiogenic cytokines The preliminary response data for 25 patients showed that 6 experienced a complete response and 4 experienced a partial response. It was previously demonstrated that the length of an initial response to platinum-based chemotherapy in ovarian cancer is predictive of the duration of future responses After coThe quality of life component of the study demonstrated little change in general and BRM-specific symptoms due to the cytokine treatments. However, both aspects of quality of life were significantly affected by carboplatin treatment. General aspects of quality of life returned to pretreatment baseline levels between treatment courses, whereas the BRM-specific symptoms remained suppressed. This difference was most likely due to the residual effects of carboplatin. Understanding the quality of life effects of novel combinations of treatment is critical. Examination of whether the BRM-specific symptoms return to pretreatment baseline levels after treatment is ended will be important to determine.In conclusion, this novel regimen of GM-CSF, IFN-γ1b, and carboplatin has an acceptable toxicity profile and significantly increases blood levels of activated MOs, platelets, WBCs, and neutrophils. Completion of the phase II study will provide data on response, time to tumor progression in responders compared with prior treatments, and toxicity and will provide an opportunity to determine whether ADCC activity recovers in responding patients.


This book on cerebrospinal fluid (CSF) proteins is primarily focused on immunoglobulins. The book was written as an extension of a meeting on multiple sclerosis to provide a more extensive consideration of the CSF. My review of this book by Dr. Thompson is written in the context of the reason I was drawn to this book and agreed to read the book and provide a review. I was drawn to the book based on its title because my research group was about to make a significant commitment to proteomic analyses of the cerebrospinal fluid (CSF) in both animal models and human disease states. I expected that, although I was familiar with CSF and the proteins that I had been studying for many years, this book would provide a foundation and reference for me and the postdoctoral fellows and students in my laboratory as we expanded our studies into the CSF. The forward indicated that the book "makes for an encyclopedia type of book for laboratory directors, experimentalists, etc". Therefore I was looking for a book on proteomics of the CSF and perhaps a state of the art assessment of protein profiling in the CSF. The first chapter of the book, a historical perspective on CSF proteins, mentions mostly IgG and associated methodologies. In Chapter 6 the discussion on pros and cons of qualitative versus quantitative analysis, a key issue in the evolving world of proteomics, is focused on IgG as the "central issue' in the studies of CSF proteins. In addition, the options for quantitative approaches do not provide much information on the strengths of ELISAs or even radioimmunoassays. The protocols are somewhat dated but in a historical context; a reasonable resource. Thus, this book is predominantly focused on immunoglobulins in the CSF with a particular focus on the oligoclonal IgG bands in multiple sclerosis. In the latter sense the book is an excellent resource and outstanding review of the field and the new investigator wishing to study immunoglobulin in the CSF can benefit from the comments of the author as a leader in this area from its beginning. As a resource itself, the book has 1,322 references. The reader interested in the broader topic of proteins in the CSF may find this book does not meet expectations.The book is divided into four segments. Part 1 has 5 chapters and deals with normal CSF proteins in a "general fashion". This section is a strength of the book and provides useful background and organizational structure for the new student of the CSF. The tables are particularly helpful, providing information on the concentrations of different proteins in the CSF relative to serum. There is a very good reference table (4.4) on percentage transfer of proteins from serum to CSF. In contrast, many of the figures are difficult to understand at best, and are overly simplistic. For example, Figure 5.10 is a cartoon showing 6 sources of CSF but the 6 sources are not labeled or otherwise identified in a figure legend.In some of the functional discussions in this section there are some questions about accuracy and some of the information is dated. Despite the abundance of references for this book, there are limited references in the discussion of the different blood and CSF barriers that should be provided to support some of the statements that invoke data as support. The function of prostaglandin synthase, induction of sleep when injected into the CSF, is confused with its end product PDG2. In another example, there is a statement that beta 2 microglobulin levels do not change in neurological disorders. Although this statement appears true for multiple sclerosis, there is certainly not uniform agreement on this in other neurological disorders -3. LaterAnother example of limited consideration of important topics is the rationale for why transferrin (Tf) without a sialic acid moiety (tau) occurs in the CSF. The treatment of this protein (and others) fails to consider the possibility that Tf made in the choroid plexus  can be rComments such as protein released into the CSF by "white matter" may be carried down into the lumbar sac but any that are released through the cerebral cortex are swept into the arachnoid villi are difficult to understand and lack evidence to support them. Another such puzzling comment is that electron microscopists had previously assumed that there was no interstitial space until more recent techniques for tissue fixation were developed. Although historically accurate, the existence of interstitial space in the brain has been known for well over 30 years  declare that they have no competing interests.Sole author


The Microfil was taken up by an extensive network of extracranial lymphatic vessels in the olfactory turbinates. In addition however, Microfil also passed consistently through the dura at the base of the brain. Microfil was noted in the spaces surrounding the venous network that comprises the cavernous sinus, in the adventitia of the internal carotid arteries and adjacent to the pituitary gland. Additionally, Microfil was observed within the endoneurial spaces of the trigeminal nerve and in lymphatic vessels emerging from the epineurium of the nerve. These results suggest several unconventional pathways by which CSF may be removed from the subarachnoid space. The movement of CSF to locations external to the cranium via these routes may lead to its absorption into veins and lymphatics outside of the skull. The physiological importance of these pathways requires further investigation.The purpose of this investigation was to enhance our understanding of cerebrospinal fluid (CSF) absorption pathways. To achieve this, Microfil  was infused into the subarachnoid space (cisterna magna) of sheep  This agent facilitates the generation of three-dimensional images of the spaces into which it has been injected. In previous reports, we infused Microfil into the subarachnoid compartment of a variety of species including mice, rats, rabbits, pigs, sheep, monkeys and humans. We noted that this contrast agent was carried through the cribriform plate into an extensive network of lymphatic vessels in the nasal submucosa of these species . These dsorption -6. Addit®  manually into the cisterna magna over 10–15 min using a syringe. In several sheep, the carotid arteries were catheterized before euthanasia and 20 ml of blue Microfil®  was infused into both arteries immediately after euthanasia. After storage overnight at 4°C, the heads were skinned, sliced in a sagittal orientation and fixed in 10% formalin. Sectioning of the fixed tissues was made in several orientations including coronal and sagittal. The tissues were viewed under a dissecting microscope  and images were captured on a Nikon digital camera (Coolpix 995). For histological analysis, the fixed tissues were sliced and embedded in paraffin. Samples were sectioned at 4 μm thickness and stained with haematoxylin and eosin. Histological assessments were performed using a Motic Digital Microscope (DMB5) and images acquired using Motic Images Advanced 3.0 software . All animal experiments were approved by the ethics committee at Sunnybrook Health Sciences Centre and conformed to the guidelines set by the Canadian Council on Animal Care and the Animals for Research Act of Ontario.Details of the methods can be found in our earlier publications ,7. BriefThe distribution of Microfil was similar in both newborn and adult sheep. Yellow Microfil was observed throughout the subarachnoid compartment associated with the base of the brain. In addition to the distribution of yellow Microfil in the lymphatic vessels in the olfactory turbinates that has been described in previous reports, a consistent finding in almost all animals that were perfused successfully, was that Microfil passed through the dura at the base of the brain and entered the cavernous sinus Figs. . The cavDuring processing for histology, the yellow Microfil turned dark brown and most of it was removed from the tissues during tissue preparation. Nonetheless, some residual Microfil was observed where it was apparently trapped by connective tissues. The yellow contrast agent was noted in the adventitia of the internal carotid arteries and was associated with the tissues around the pituitary gland Figs. . The spapost mortem. Nonetheless, Microfil distribution patterns have proven to be useful in the past to elucidate lymphatic CSF absorption pathways and might provide insights into several other 'unconventional' pathways by which CSF might exit the subarachnoid compartment to reach extracranial sites. These potential routes include CSF entry into dura, CSF passage along the adventitia of blood vessels, CSF transport within nerves and CSF movement through dura into the peri-venous spaces associated with the cavernous sinus.We recognize the need to be cautious in extrapolating physiological concepts from anatomical studies employing Microfil, particularly because the Microfil is infused Regarding the movement of CSF into dura, we observed that yellow Microfil passed through the dura at the base of the brain although in this case, it seemed to do so only in association with the cuff of adventitia of the internal carotid artery. In contrast, in a previous report we noted that yellow Microfil coloured the dura proper in the vicinity of the superior sagittal sinus . If the There is however, at least one theoretical objection to a possible role for dural lymphatics in CSF drainage. The cellular architecture and the presence of tight junctions between arachnoid cells are believed to contribute to the blood-brain/CSF barrier . WithoutThere is support for the view that parenchymal interstitial fluid or CSF can be carried through the adventitia of cerebral blood vessels -18. It wThe fact that CSF can move along the outer surface of nerves as they leave the brain and spinal cord is well established in the literature ,7,19,20.In the literature, there is some support for the concept that the CSF compartment and extracranial lymphatic vessels are connected by fluid-filled spaces within the nerves. For example, horseradish peroxidase injected intrathecally was observed in epineural, perineural and endoneural spaces and extended beyond the epineurium into lymphatics . In addiOne of the most consistent findings was the appearance of Microfil in the peri-venous spaces of the cavernous sinus. The existence of the contrast agent at this location may have been due to movement through the dura, passage along the adventitia of blood vessels, or possibly associated with transport along the various nerves which enter and leave this space. To the best of our knowledge, there have been no previous reports of CSF movement into the cavernous sinus.The complex formed by the superior sagittal sinus and arachnoid villi and granulations is the most well studied interface between CSF and venous blood. Absorption of CSF from the subarachnoid space across the arachnoid projections into the sinus is assumed to be driven by the hydrostatic pressure difference between the two compartments. Blood vessels and nerves do not transect the dura of the superior sagittal sinus (as they do the cavernous sinus). Accordingly, perivascular and endoneurial spaces are not part of the hypothesized CSF absorption system at this location.While there has been a report of the presence of arachnoid projections in the cavernous sinus, their physiological significance has never been elucidated . HoweverIn summary, the presence of intracisternally-administered Microfil external to the dura at the base of the brain suggests several ways in which CSF may gain access to potential absorption sites. The interstitium of the dura, adventitia of blood vessels and endoneurium of nerves might provide a link between the subarachnoid space and extracranial tissues. However, unless evidence is obtained to the contrary, it seems likely that these 'conduits' are of only minor significance to the volumetric clearance of CSF from the brain. From a volumetric perspective, absorption of CSF into the veins of the cavernous sinus would appear to have greater potential, a concept that appears to warrant further investigation.The author(s) declare that they have no competing interests.MJ: conceived of the study, and participated in its design and coordination.DA: assisted in all experiments and helped in the preparation of the manuscript.LK: helped in the preparation of the manuscript.All authors read and approved the final manuscript.


Despite cost containment efforts, parenteral (IV) ciprofloxacin appears to be overutilized at Vancouver General Hospital. In November 2003, the Pharmacist-managed intravenous to oral (IV-PO) Dosage Form Conversion Service was implemented, enabling autonomous pharmacist-initiated dosage form conversion for ciprofloxacin. This study evaluates characteristics of ciprofloxacin use prior to and following implementation of this conversion service.This was a single-centre, two-phase (pre/post), unblinded study. Phase I occurred between November 12, 2002 and November 11, 2003 (365 days), and Phase II between November 12, 2003 and March 11, 2004 (120 days). All patients receiving ciprofloxacin IV during these periods were reviewed. The primary endpoint was IV:PO ciprofloxacin use ratio. Secondary endpoints were total number of ciprofloxacin doses, proportion of inappropriate IV ciprofloxacin doses, cost of therapy between phases, and estimated cost avoidance with the intervention.Two hundred ciprofloxacin IV treatment courses were evaluated (100 per phase). The IV:PO ciprofloxacin use ratio was 3.03 (Phase I) vs. 3.48 (Phase II). Total number of doses and ratio of IV to total doses across phases were similar (p = 0.2830). IV-PO ciprofloxacin conversion occurred in 27/100 (27%) of IV courses in Phase I and 23/100 (23%) in Phase II. Proportion of inappropriate ciprofloxacin IV doses decreased between Phases I and II (244/521 (47%) vs. 201/554 (36%) (p = 0.0005), respectively). Furthermore, the proportion of pharmacist-preventable inappropriate ciprofloxacin IV doses was reduced between Phases I and II (114/244 (47%) vs. 65/201 (32%) (p = 0.0026). Proportional cost avoidance associated with total inappropriate IV use was $7,172/$16,517 (43%) (in Canadian dollars) in Phase I vs. $6,012/$17,919 (34%) in Phase II (p = 0.001). Similarly, proportional cost avoidance associated with pharmacist-preventable inappropriate IV doses was reduced from $3,367/$16,517 (20%) in Phase I to $1,975/$17,919 (11%) in Phase II (p = 0.001).While overall utilization of ciprofloxacin remained unchanged and the proportion of IV to total doses was stable during the study period, the proportion of inappropriate IV doses and its associated costs appear to have declined subsequent to implementation of a Pharmacist-managed IV-PO Dosage Form Conversion Service. Such a program may be a beneficial adjunct in facilitating appropriate and cost-effective usage of ciprofloxacin. The annual drug expenditures at Vancouver Hospital are approximately $13 million (in Canadian dollars). At the Vancouver General Hospital (VGH) site, anti-infectives accounted for an expenditure of $3.39 million or 25% of the 2002–03 fiscal year total drug costs. Ciprofloxacin ranked third among all drugs by cost and was the second highest annual expenditure within the anti-infective drug class at $646,000. In addition, ciprofloxacin expenditures increased 5% from the previous year. Of the 39,147 ciprofloxacin doses administered in the 2002–03 fiscal year, 18,297 doses (47%) were given intravenously (IV).Ciprofloxacin is a fluoroquinolone antibacterial that is primarily active against aerobic gram-negative bacterial infections . It can In an effort to optimize use and minimize drug expenditures, ciprofloxacin has been designated a reserved antimicrobial drug (RAD) at our institution and has been included in the existing intravenous to oral (IV-PO) Step-down Program since 1992 . Under tDespite these cost containment efforts, there is some evidence to suggest that the IV formulation of these drugs may not be optimally utilized. Previous investigations by others and ourselves have shown that the IV formulation is often initiated when the PO formulation can be used -13. Of eVarious authors have described criteria-based medication dosage form conversion programs in the literature ,13,15-24In November 2003, the Pharmacist-managed IV-PO Dosage Form Conversion Service was approved at VGH by the Antibiotic Use Subcommittee (AUS), Drugs and Therapeutics Committee (D&TC), and the Medical Advisory Committee (MAC). Several antimicrobial agents were included in this service; namely, ciprofloxacin, clindamycin, co-trimoxazole, fluconazole, levofloxacin, metronidazole, acyclovir, ampicillin, cefazolin, cefuroxime, penicillin G, ceftriaxone, imipenem-cilastatin, cloxacillin, erythromycin, and ticarcillin-clavulanate .Our hypothesis was that ciprofloxacin IV was overutilized at VGH and that a more cost-effective use of this dosage form was possible with the implementation of a Pharmacist-managed IV-PO Dosage Form Conversion Service. Accordingly, this study was conducted to assess the impact of this service on the relative utilization of the IV versus PO dosage form of ciprofloxacin. To our knowledge, there are no published reports involving an assessment of the impact of a Pharmacist-managed IV-PO Dosage Form Conversion Service on ciprofloxacin usage characteristics at a major Canadian teaching hospital.A literature search of the Medline, EMBASE and IPA databases, as well as a bibliographic review from the cited articles was performed to retrieve references pertaining to pharmacy-managed IV-PO conversion programs. Additional references were obtained through the St. Paul's Hospital and Lions' Gate Hospital pharmacy departments, as these local institutions had established pharmacist-managed conversion services ,23. The This was a single-centre, 2-phase (pre/post), unblinded study to assess the impact of a hospital-approved intervention aimed at improving the utilization of ciprofloxacin dosage forms. Phase I  was designed to characterize ciprofloxacin usage patterns under the existing IV-PO Step-down Program. Phase II  was designed to characterize the impact of the new Pharmacist-managed Dosage Form Conversion Service  on the relative utilization of the ciprofloxacin IV and PO dosage forms.The primary endpoint was the relative utilization of IV and PO ciprofloxacin by dose (IV:PO ciprofloxacin use ratio). The secondary endpoints were the overall utilization of ciprofloxacin (by dose), the proportion of total IV doses considered to be inappropriate, and relative IV and PO total and treatment course acquisition costs between the two phases. Potential cost avoidance associated with the intervention was estimated from the data.Prior to the implementation of the program, pharmacists were educated on the approved conversion service through in-house presentations. A newsletter was distributed to all medical staff detailing the program .Decentralized clinical pharmacists on the medical wards were expected to conduct target drug report reviews 5 days per week to identify inpatients who had been prescribed ciprofloxacin IV. Health records were then reviewed, and patients assessed to determine if IV-PO conversion criteria were met. A patient was eligible for IV-PO dosage form conversion after 48 hours of IV therapy if he/she 1) continued to need an antibiotic; 2) was clinically stable; 3) was capable of tolerating the PO dosage form; and 4) had no factors present that would adversely affect PO bioavailability . Pharmacists could consult the Infectious Diseases service or Infectious Diseases Pharmacist at anytime with any questions regarding IV-PO conversion eligibility.For patients who met the conversion criteria, the pharmacist would write the order for the PO regimen in the Physician's Orders section of the health record. If the pharmacist wanted to convert the patient to PO ciprofloxacin prior to 48 hours of IV therapy, they would first confer with the physician. In collaboration with the healthcare team, the pharmacist would monitor the patient for clinical progress and medication tolerability, and could convert the patient back to IV therapy as required.A randomly selected convenience sample of 200 ciprofloxacin IV treatment courses (100 treatment courses per phase) was considered to be adequate to determine the impact of this new intervention. These courses were identified using computerized pharmacy records and a random sample was undertaken using a computer-generated number list.All patients who were ordered ciprofloxacin IV were included in the sample collection. Patients were excluded if they did not receive any doses of ciprofloxacin IV, if their ciprofloxacin IV treatment course did not occur within the pre-specified phase to which they were randomized, or if their charts were unavailable from the Health Records department at VGH as of July 14, 2004.Charts were reviewed to gather demographic data, ciprofloxacin utilization information, inappropriate IV doses, and pharmacist-preventable IV doses of ciprofloxacin.© Version 11.0). Any treatment course that this investigator considered to have four or more inappropriately administered ciprofloxacin IV doses was reviewed in collaboration with the coordinating investigator to ensure accuracy of interpretation.Data was collected by one investigator and entered into statistical analysis software . The inappropriate IV-PO ciprofloxacin acquisition cost was the differential cost between the IV and the PO dosage form at current contract prices multiplied by the number of inappropriate IV doses administered.Two hundred and fifteen health records of patients who were prescribed ciprofloxacin IV during the study period were reviewed. Of these, seven patients were excluded, as the ciprofloxacin IV treatment courses were not completed within the pre-specified treatment phase. Six patients were excluded, as no IV ciprofloxacin doses were actually received. Health records were not accessible at the time of the study for the remaining two patients. Accordingly, 200 treatment courses for 200 patients (100 per phase) were included for analysis. This represented a 4% (100/2411 treatment courses) sampling rate for Phase I and a 10% (100/994 treatment courses) sampling rate for Phase II.Patient demographics are presented in Table Of the 200 ciprofloxacin IV courses reviewed, the total number of doses and the ratio of IV to total doses across phases were similar (p = 0.2830) Figure . The IV:Ciprofloxacin treatment characteristics are described in Table For those patients who met the criteria for the use of an oral dosage form, 59/100 (59%) received one or more inappropriate doses of ciprofloxacin IV in Phase I compared to 61/100 (61%) in Phase II. There was a significant decrease in the proportion of inappropriate ciprofloxacin IV doses between phases (244/521 (47%) in Phase I vs. 201/554 (36%) in Phase II (p = 0.0005)  of total ciprofloxacin costs in Phase I and $17,919 (98%) of these costs in Phase II  of ciprofloxacin doses deemed inappropriate and preventable by pharmacists were still administered. Ideally, all of these doses should have been avoided. One full working day (excluding weekends and statutory holidays) was allotted as the time required for clinical pharmacists to assess these patients. This delay in IV-PO conversion may be explained in part by having our data collection period immediately after the introduction of the new service, as pharmacists may not yet have been comfortable exercising a dosage form conversion autonomously. The retrospective assessment for appropriateness by the investigator may also differ from that of the clinical pharmacist. It would be beneficial to obtain an internal assessment to discover the barriers associated with the program. Of course, it would be preferable to educate the medical staff to initiate PO regimens where indicated and avoid the use of the IV formulation.Several limitations exist with this study. The retrospective, pre/post, unblinded design precludes the formulation of any direct causal relationships between the implementation of the conversion service and the subsequent reduction in inappropriate ciprofloxacin IV doses. The sample size of convenience may not have achieved the power required to detect a difference. In addition, the sampling rate was relatively low at 4.1% (100/2411 ciprofloxacin IV courses) in Phase I and 10.1% (100/994) in Phase II, and thus may not truly represent the characteristics of our population. Time restrictions resulted in differences in sampling periods between Phase I (365 days) and Phase II (120 days), which may affect the representation of ciprofloxacin IV treatment courses throughout the year. However, this should not directly influence the proportion of inappropriate and pharmacist-preventable inappropriate ciprofloxacin IV treatment doses. The assessment of this service was over a relatively short period, and so may not truly reflect the long-term impact of this program.As with any unblinded study of this type, the potential for investigator assessment bias existed. As the evaluation of dose appropriateness was undertaken sequentially across phases, potential bias may have been introduced as the investigator gained experience during the process. To minimize this bias, charts were reviewed by a senior investigator when greater than four inappropriate and pharmacist-preventable inappropriate ciprofloxacin IV doses were identified by the junior investigator. A 100% concordance existed between the assessments made by the junior and senior investigators.In the context of drug use optimization and cost minimization, implementation of a Pharmacist-managed IV-PO Dosage Form Conversion Service may be used to facilitate appropriate, cost-effective therapy. This service can be used in conjunction with other established methods including newsletters , chart tIn summary, the overall utilization of ciprofloxacin seems to have remained unchanged and the proportion of IV to total doses appears stable. However, the proportion of inappropriate IV doses and its associated costs appear to have declined subsequent to the implementation of a Pharmacist-managed IV-PO Dosage Form Conversion Service. Such a program may be a beneficial adjunct in facilitating appropriate and cost-effective usage of ciprofloxacin.The author(s) declare that they have no competing interests.BPH participated in the design of the study, performed data collection and analyses, and drafted the manuscript. TTYL participated in the design and coordination of the study, performed data and statistical analyses, and drafted and revised the manuscript. RMB participated in the design of the study, developed the analytical database, and revised the manuscript. TLN participated in the design of the study and revision of the manuscript. PJJ conceived the study, participated in its design, performed data and statistical analyses, and drafted and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We generated the gene expression profile of the total testis from the adult C57BL/6J male mice using serial analysis of gene expression (SAGE). Two high-quality SAGE libraries containing a total of 76 854 tags were constructed. An extensive bioinformatic analysis and comparison of SAGE transcriptomes of the total testis, testicular somatic cells and other mouse tissues was performed and the theory of male-biased gene accumulation on the X chromosome was tested.We sorted out 829 genes predominantly expressed from the germinal part and 944 genes from the somatic part of the testis. The genes preferentially and specifically expressed in total testis and testicular somatic cells were identified by comparing the testis SAGE transcriptomes to the available transcriptomes of seven non-testis tissues. We uncovered chromosomal clusters of adjacent genes with preferential expression in total testis and testicular somatic cells by a genome-wide search and found that the clusters encompassed a significantly higher number of genes than expected by chance. We observed a significant 3.2-fold enrichment of the proportion of X-linked genes specific for testicular somatic cells, while the proportions of X-linked genes specific for total testis and for other tissues were comparable. In contrast to the tissue-specific genes, an under-representation of X-linked genes in the total testis transcriptome but not in the transcriptomes of testicular somatic cells and other tissues was detected.Our results provide new evidence in favor of the theory of male-biased genes accumulation on the X chromosome in testicular somatic cells and indicate the opposite action of the meiotic X-inactivation in testicular germ cells. Except for the characterization of the total testis transcriptome, we compared our data with the publicly available SAGE library from adult testis somatic cells [In the present work we used Serial Analysis of Gene Expression (SAGE)  to charaA/J  and C57Bic cells  and othe2 = 0.84 for all unique tags, Pearson correlation), which suggests a good reproducibility of SAGE data. However, a certain variation was observed in the tag abundances when 24 529 unique tags found in both total testis SAGE libraries were compared by Monte Carlo simulations. Three hundred thirteen tags exhibited significant differences in their frequency between TT 1 and TT 2 libraries at p < 0.05 (89 tags at p < 0.01) representing non-hereditary variations in transcription profiles and variations introduced by the experimental process. The fold factor value  for 93.5 % of the compared tags ranged between -2.2 and 2.2 (for 99% of the tags between -5 and 5). Dot plot comparison and fold factor distribution graphs  displayed two tags separated by 87 bp in mt-Co3 gene mRNA. One isoform was predominantly present in the ATSC library and the other was observed exclusively in the TT 1+2 library. Substantial over-expression of mitochondrial cytochrome c oxidase complexes I, II, III and NADH dehydrogenase 3 and 4 was noted in testicular somatic cells  and exhibited at least fivefold enrichment of tag counts (fold factor <-5 or > 5). According to this criterion a set of 829 genes is expressed predominantly in germ cells and 944 genes are expressed mainly from the somatic part of the testis see . Moreoveells see .We examined the similarity of B6 testis transcriptomes to other available mouse SAGE transcriptomes created from normal and diseased bulk tissues by hierarchical clustering. Thirty-two SAGE libraries containing 190 871 unique tags (including single copy tags) were used as input in this analysis see . We compPrevious works have shown a significant enrichment of prostate- and spermatogonia-specific genes on the X chromosome when compared to autosomes ,30. We a-6, Chi-square test). We can conclude that the number of expressed X-linked genes is underrepresented in the transcriptome of total testis.Out of the 14 222 genes expressed in SAGE libraries from total testis, adult testis somatic cells and 7 somatic tissues  see  we consiThe same set of 14 222 genes was examined for the distribution of tissue-specific genes on autosomes and the X chromosome. We compared the genes specific for either total testis Table  or adultBased on the data from testis and other publicly available SAGE libraries see  we identFurther we analyzed the genome organization of genes preferentially expressed in testis. We evaluated the expression of 14 222 genes among the studied tissues and for 12 331 genes we were able to assign a genomic position according to the NCBI mouse genome assembly . The genomic position was resolved for 5 252 and 5 843 genes expressed in total testis and testicular somatic cells, respectively, including 1 438 (27.4%) and 1 197 (20.5%) preferentially expressed genes, respectively see . To evalRsaI) used in construction of the libraries and the limited availability of data from the BDF1 testis transcriptome, we could perform only a rough manual comparison of our B6 testis transcriptome (76 854 tags) and the combined BDF1 testis transcriptome from 3- and 29-month-old BDF1 mice (41 221 tags). We focused on the most highly expressed testicular genes in GNF Mouse Atlas v2 [Serf1 gene could not be distinguished because its low complexity tag matches multiple genes and the Cox7a2 gene is not detected because its transcript lacks NlaIII restriction site). In contrast, only 9 genes were detected in the BDF1 testis library, 13 genes were missing due to the absence of RsaI restriction site in the transcript and for 13 other genes the expression data from BDF1 testis were not publicly available. Furthermore, out of the 35 highly expressed genes in testis, 21 genes were among the top 100 most expressed genes in the B6 total testis library, but only 9 genes were among the top 100 most expressed genes in the BDF1 total testis library. It appears that our SAGE data from the B6 testis transcriptome shows better correspondence to the microarray data than the data from the transcriptome of BDF1 testis.In a recent study focused on senescence changes in testis, a modified SAGE method was used to generate digital gene expression profiles of total testis from 3- and 29-month-old mice of the BDF1 strain and 14-month-old mice of the SAMP1 strain that exhibits an accelerated senescence . BecauseAtlas v2 ,34 that Serial analysis of gene expression is a high-throughput method for building a catalogue of expressed genes and their expression levels of "normal" as well as diseased or genetically variant tissues and organs . The digIn this study we constructed a SAGE library of the total testis of the C57BL/6J (B6) mouse inbred strain, compared it with other public available mouse SAGE libraries and analyzed localization of testis-expressed genes within the mouse genome. The B6 strain was favored for the availability of its high-quality draft genomic sequence  and becaContrary to microarrays, SAGE data are platform independent, which permits the use of unrelated datasets coming from various sources to compare gene expression patterns. We analyzed the mouse testis transcriptome by comparing our total testis SAGE library to the adult testis somatic cells library  and to aCaenorhabditis elegans [Drosophila [Conflicting results have been reported on the representation of male-biased genes on the X chromosome in various species. Spermatogonia-specific genes were found to be an order of magnitude more abundant on the mouse X chromosome . In huma elegans  and in Dosophila ,43. In tosophila -7. Our posophila -46. ThusTestis-specific genes belong to a wider category of sex-biased genes, which according to the hypothesis of sexually antagonistic genes are more likely to spread on the X chromosome than on autosomes . This isDrosophila and humans [Drosophila melanogaster one third of testes-specific genes occur in clusters [The eukaryotic gene order is nonrandom obviously not only due to shifting of sex-biased genes to and from the X chromosome, but also owing to a nonrandom clustering of genes within chromosomes. This somewhat unexpected conclusion (taking into account the relative autonomy of transgene regulation) is gaining gradual support from global transcriptome analyses of various eukaryotic species  . The obsd humans ,49, clusd humans , clusterd humans  or genesd humans . In Drosclusters , a phenoclusters  to definWe identified chromosomal clusters of adjacent genes with preferential expression in testis that contain a significantly higher number of genes than expected by chance. This phenomenon is not merely a consequence of tandem duplication. The genes with specific expression in testicular somatic cells are more abundant on the X chromosome, which favors the theory of accumulation of male-biased genes on the X chromosome. In contrast, the X-linked genes are under-represented in the transcriptome of total testis, which is in accordance with the idea of X-chromosome inactivation during the first meiotic division.Mice were housed in specific pathogen free environment and their manipulation was in accordance with the Czech Animal Protection Act No. 246/92, 162/93, and decrees No. 311/97, fully compatible with the NIH Publication No. 85-23, revised 1985. Testes were obtained from 9-week-old males of the C57BL/6J mouse strain. The animals were killed by cervical dislocation; the testes were quickly removed from the body and released from tunica. The total RNA was extracted from homogenized testes using TRIzol (Invitrogen) according to the manufacturer's protocol. SAGE libraries were constructed from the total RNA isolated from both testes of a single male (TT 1) and from the pool consisting of equal weight amounts of total RNA isolated from both testes of three male littermates (TT 2).NlaIII as the anchoring enzyme and BsmFI as the tagging enzyme. Two minor modifications of the MicroSAGE protocol were employed: the first strand cDNA synthesis reaction was incubated at 42°C and the amount of linkers used in the linker ligation step was decreased to ~10 ng. Sequencing was performed in a Beckmann Coulter CEQ 2000 DNA Analysis System. The sequence files were processed for the tag extraction using a custom Perl script. Tags were extracted only from clones containing > 2 ditags. Duplicated ditags, linker tags and all 1-bp linker variations were removed. Data of total testis SAGE libraries are available in the GEO repository [SAGE libraries were constructed as described in the MicroSAGE protocol version 1.0e available from SAGE homepage  using NlTag identification to UniGene clusters was done using three methods: SAGEmap reliable mapping , Mouse STags significantly different between SAGE libraries were determined by Monte Carlo simulations. Using the described algorithm  a set ofThe SAGE library from somatic cells of the adult testis  was obtaThirty-two mouse SAGE libraries constructed from bulk tissues  that were publicly available to date  were selected see . For eacTwenty-seven SAGE libraries created from bulk tissues (excluding tumors) were organized into 7 groups by tissue type and tag counts from SAGE libraries within each group were combined see . The groAnalysis was done in parallel for testis-specific genes in total testis and somatic cells of adult testis. The tissue-specific genes were selected according to tag counts in the testis tissue and 7 non-testis tissues see . A gene TT) and adult testis somatic cells (PEMATSC) were calculated for each gene. The gene was considered to be preferentially expressed in total testis if PEMTT> = 1/2 PEMTT(max), and in somatic cells of adult testis if PEMATSC> = 1/2PEMATSC(max). PEM(max) values represent the maximum PEM value encountered in the tissue, PEMTT(max) = 1.169, PEMATSC(max) = 1.145.The preferential expression measure (PEM)  was used-10 and with an alignment of at least 50% length and 30% identity of the query sequence were processed and identified to LocusLink genes. If a LocusLink gene located in the vicinity of the original LocusLink gene was found among the hits (considering 10 adjacent genes in both directions), both genes were considered as a tandem duplicate pair and were excluded from the genome. As a result a tandem duplicate-free genome with 16 858 LocusLink genes was obtained.To prepare a tandem duplicate-free mouse genome we considered 19 684 known genes from the LocusLink database that were mapped on the mouse genome assembly (NCBI build 32) . For eacTwo sets of gene clusters with preferentially expressed genes were identified – for total testis and somatic cells of adult testis. All LocusLink genes from the tandem duplicate-free mouse genome were associated with the expression status . Each chromosome was searched using a sliding window of three adjacent genes and three consecutive preferentially expressed genes were considered as a cluster (tight clusters). Another search was performed using a sliding window of six adjacent genes and at least three preferentially expressed genes were required to form a cluster spanning from the first to the last preferentially expressed gene (loose clusters). The overlapping clusters were merged into a single cluster encompassing all involved genes (separately for tight or loose clusters). The permutations performed 100 000 random shufflings of the expression status in the genome while keeping the gene positions constant. A search with the above defined sliding windows determined the number of preferentially expressed genes located in clusters in each randomized genome. The p-value (one tailed) was computed as the number of permutations yielding greater than or equal to the observed number of preferentially expressed genes located in clusters.All statistical analyses, including Monte Carlo simulations, hierarchical clustering, chromosomal and gene permutations were conducted in R statistical environment  using cuThe following database versions were used in all analyses: Mouse UniGene build #136 , mouse SAGEmap  corresponding to the mouse UniGene #136, LocusLink , mouse genome assembly NCBI build 32 (November 2003), mouse Reference Sequence collection  and Gene Ontology database .PD constructed the SAGE libraries and performed the bioinformatic analyses. CV carried out sequencing of SAGE libraries. PS participated in bioinformatic analysis of gene order. VP is Head of the Center for Integrated Genomics. JF conceived the study and coordinated work. PD and JF wrote the article. All authors read and approved the final manuscript.SAGE tags detected in mouse total testis. List of 7 481 tags with tag count > 1 in combined total testis SAGE library with reliable tag identification according to the Mouse SAGE Site database.Click here for fileTags with significantly different tag counts between total testis and adult testis somatic cells. List of 3 258 tags with significantly different tag counts between the total testis and adult testis somatic cells SAGE libraries determined by Monte Carlo simulations . The reliable tag identification according to the Mouse SAGE Site database is provided.Click here for fileGenes with predominant expression in germinal and somatic cells of the testis. List of 924 and 802 genes with predominant expression in germinal and somatic cells of the testis, respectively, based on the comparison of total testis and adult testis somatic cells SAGE libraries. Tags with significantly different tag counts  and at least five-fold increased/decreased tag counts were selected and identified using RNA evidence mapping.Click here for fileMouse SAGE libraries used in genomic analysis and hierarchical clustering. List of mouse SAGE libraries publicly available to date July 1, 2004 that were used in genomic analysis and hierarchical clustering.Click here for fileGenes specific for total testis or adult testis somatic cells. List of 395 and 81 genes with specific expression in total testis or adult testis somatic cells determined by comparison to SAGE data from seven non-testis tissues . A gene was considered to be testis specific if the corresponding tags were present only in total testis or adult testis somatic cells SAGE libraries and missing in all non-testis libraries. Tags were identified using RNA evidence mapping.Click here for fileTestis expressed genes located on the X chromosome (summary). Summary of the X-linked genes expressed in total testis and adult testis somatic cells. Tags were identified using RNA evidence mapping.Click here for filePreferentially expressed genes in total testis and testicular somatic cells. Preferentially expressed genes were determined separately for total testis and adult testis somatic cells in conjunction with their expression in seven non-testis tissues. Expression of genes was scored using preferential expression measure (PEM). A gene was considered to be preferentially expressed if PEM score was above 50 % of the maximum PEM value encountered in that tissue. Tags were identified using RNA evidence mapping.Click here for fileGenes preferentially expressed in testis located in chromosomal clusters within tandem duplicate-free genome. Chromosomal clusters of genes preferentially expressed in testis were localized by the search with a sliding window. Two types of clusters were identified: tight clusters  and loose clusters .Click here for fileManual comparison of the most highly expressed genes in three total testis transcriptomes . The list of 35 most highly expressed genes in total testis according to the GNF Mouse Atlas v2 organized with the appropriate NlaIII and RsaI SAGE tags extracted from their representative mRNA/RefSeq sequences.Click here for file


Central nervous system (CNS) exposure to HIV is a universal facet of systemic infection. Because of its proximity to and shared barriers with the brain, cerebrospinal fluid (CSF) provides a useful window into and model of human CNS HIV infection.Prospective study of the relationships of CSF to plasma HIV RNA, and the effects of: 1) progression of systemic infection, 2) CSF white blood cell (WBC) count, 3) antiretroviral therapy (ART), and 4) neurological performance. One hundred HIV-infected subjects were cross-sectionally studied, and 28 were followed longitudinally after initiating or changing ART.10 copies/mL). CSF HIV viral loads (VLs) correlated strongly with plasma VLs and CSF WBC counts. Higher CSF WBC counts associated with smaller differences between plasma and CSF HIV VL. CSF VL did not correlate with blood CD4 count, but CD4 counts <50 cells/μL associated with a low prevalence of CSF pleocytosis and large differences between plasma and CSF VL. CSF HIV RNA correlated neither with the severity of the AIDS dementia complex (ADC) nor abnormal quantitative neurological performance, although these measures were associated with depression of CD4 counts.In cross-sectional analysis, HIV RNA levels were lower in CSF than plasma (median difference 1.30 logIn subjects starting ART, those with lower CD4 counts had slower initial viral decay in CSF than in plasma. In all subjects, including five with persistent plasma viremia and four with new-onset ADC, CSF HIV eventually approached or reached the limit of viral detection and CSF pleocytosis resolved.response to or a contributing cause of CSF infection remains uncertain. Slowing in the rate of CSF response to ART compared to plasma as CD4 counts decline indicates a changing character of CSF infection with systemic immunological progression. Longer-term responses indicate that CSF infection generally responds well to ART, even in the face of systemic virological failure due to drug resistance. We present simple models to explain the differing relationships of CSF to plasma HIV in these settings.CSF HIV infection is common across the spectrum of infection and is directly related to CSF pleocytosis, though whether the latter is a  Frequent abnormalities in cerebrospinal fluid (CSF), including increased white blood cells (WBCs), were recognized early in the AIDS epidemic, not only in patients examined toward the end of their course who suffered neurological complications , but alsIn order to better interpret CSF findings, it is essential to understand what factors contribute to elevated CSF HIV RNA concentrations. How do systemic infection and its progressive damage to the immune system affect the VL in a non-lymphatic compartment like the CSF space? How does CSF infection respond to ART? What is the origin and importance of the CSF cell reaction detected as CSF lymphocytic pleocytosis and how does this cell reaction respond to ART? What is the effect of ART on neurological function in subjects presenting with ADC?To address these questions and better understand the relationship of the CSF HIV RNA levels to other aspects of infection and clinical findings, we undertook a prospective study of CSF in a broad range of HIV-infected subjects using two complimentary approaches. The first involved cross-sectional analysis of a clinically diverse subject sample. The second longitudinally followed subjects initiating ART. In addition to its direct clinical implications, this longitudinal approach used treatment as an 'experimental' intervention to dissect dynamic aspects of the relationships among these study variables.One-hundred subjects were entered into these studies between November, 1996 and June, 2001 in the context of protocols approved by the University of California, San Francisco (UCSF) Committee on Human Research (CHR); follow-up on a few continued until December 2004. Informed consent was obtained from all subjects. In the case of one subjects with ADC, consent was also obtained from his sister with durable power of attorney. Subjects were excluded if they suffered HIV-related or other active CNS diseases except ADC. The cross-sectional analysis targeted a total of 100 subjects, including the baseline observations of subjects starting treatment, along with a previously-reported group who stopped treatment ,15. Subjde novo or representing a change of drugs) were prescribed by subjects' primary care-giver or determined by another clinical trial. Exceptions were five subjects who entered an open-label study of high-dose abacavir (600 mg twice daily) which was completed before abacavir was licensed for clinical use [Treatment decisions were independent of this CSF study. New therapies  cpm for values below the detection limit. Concurrent paired CSF and plasma samples were treated identically and run at the same time. HIV RNA concentrations were transformed to log10 values for all analysis. Because limited serial sampling during the acute phase of treatment-induced viral decay precluded complex modeling, estimates of acute-phase HIV-RNA half-lives were derived by assuming simple exponential decay during an initial phase (days 0 – 11) as previously described [10 HIV-RNA values. The subject's acute-phase HIV-RNA half-life was log102/λ.HIV RNA was measured in cell-free CSF and plasma by the Roche Amplicor Monitor assay  using the standard and Ultrasensitive extraction methods. The latter has a quantitation limit of 50 and a detection limit of approximately 20 HIV RNA cpm. We used results in the range of 20 – 50 cpm for data reporting and analysis, and assigned a default 'floor' value of 19 (logescribed . While t50) relative to a wild-type virus reference standard, while genotypic analysis compared RT and PR sequences in the blood or CSF HIV populations to the reference wild-type .Antiretroviral drug resistance was assessed by both phenotypic and genotypic methods in selected subjects using the PhenoSense™ HIV assay  to analyze functional susceptibility in a recombinant assay and mutations in the reverse transcriptase (RT) and protease (PR) regions . Phenotyp-values were two-sided, with p-values < 0.01 considered significant. Statistics were performed using SPSS 11.5 , or Prism 4.0 .Because of the skewed distributions of several study variables, unless otherwise indicated, the median and interquartile range  were used for descriptive statistics, and nonparametric tests were used for comparisons. In the cross-sectional analysis, all cross-sectional only group and studied only once, 26 were in a longitudinal treatment group and were followed with serial LPs, and nine subjects were in a longitudinal Structured Treatment Interruption (STI) group and are reported elsewhere [treatment failures, as defined by plasma HIV RNA concentration >500 RNA cpm.Of the 100 subjects in the cross-sectional study, 65 were recruited into a lsewhere ,15. HoweReflecting the demography of the local epidemic, over 90 percent of our subjects were men, with a median age of 39 years. The majority of the cross-sectional only sample was on ART, though VL was undetectable in plasma in only eight of these 65 subjects, reflecting a bias toward entry of subjects with virological failure.While most of the 28 subjects in the longitudinal treatment group were ART-naive or had limited prior therapy, seven were on therapy, changing or adding one or more drugs at baseline. Twenty-four of 80 subjects without confounding neurological conditions (30%) were diagnosed with ADC: 13 subjects with ADC Stage 1, 8 subjects with ADC Stage 2, and 3 subjects with ADC Stage 3. Most had stable neurological impairment and clinically 'inactive' CNS disease (exceptions are discussed below). The prevalence of ADC was the principal reason for the overall median QNPZ-4 score below "normal".ΔPlasma:CSF (log10 plasma HIV RNA – log10 CSF HIV RNA), varied widely, ranging from -1.32 to 4.08 log10 cpm . More notably, the median CSF values differed by more than 10-fold (p = 0.001), and therefore the median ΔPlasma:CSF of the failure group was greater than the median for subjects off treatment. This post hoc analysis raises the question of whether 'failed' treatment might alter the relationship of CSF to plasma HIV, and indicates that despite plasma HIV escape, treatment still had an effect on CSF.The treated subjects had lower VLs in both compartments . In contrast, CSF HIV did not correlate with the blood CD4+ cells, ADC stage or QNPZ-4 scores. However, as shown on Figure 10 cpm with IQR 4.67 – 5.57, while the median plasma VL of the remaining 75 subjects was 4.35 log10 cpm with IQR 2.92 to 4.98 (p = 0.001). In contrast, the CSF VLs of subjects in this first quartile  did not differ from those of the remaining 75 subjects  (p = 0.650). The combined effect of higher plasma VL and similar CSF VL in the first quartile compared to the other subjects resulted in a greater median ΔPlasma:CSF for subjects in the first quartile , relative to the median for the remaining 75 subjects  (p = 0.001).Overall, CSF HIV RNA correlated with only two other variables examined, the plasma VL and the CSF WBC count , composed of 85–100 percent lymphocytes, with the remainder mononuclear cells. All 24 were asymptomatic, despite a median count of 15 cells/μL . The CSF WBC count did not significantly correlate with plasma VL.both pleocytosis and high plasma VLs . These were also the subjects with the highest CSF VLs. By contrast, many subjects with similarly high plasma VLs without pleocytosis had lower CSF VLs. Three subjects with CSF VLs that substantially exceeded those of plasma are indicated by subject number in Figure 10 cpm (IQR -0.115 – 0.425 copies) – far below the difference seen in the group overall.Figure p = 0.01; rho 0.254), Figure p = 0.009; t-test).CSF WBC counts showed only a modest correlation with CD4 counts , and both also correlated with the CD4 count . For all ADC subjects, the CD4 median was 67.5 cells/μL (IQR 28.0 – 124.5); for the untreated ADC subgroup, the median was 35 (IQR 14.5 – 67.5) cells/μL, and for those on ART, the median was 95 cells/μL (IQR 36 – 205).Neither the ADC stage nor the QNPZ-4 score correlated with the CSF or plasma VLs across the cross-sectional group, though these two measures correlated strongly with each other . Figure 10 HIV RNA values in CSF and plasma [The graphs in Figure d plasma , and resp < 0.000, rho -0.744), and that neither the baseline VLs themselves, the CSF WBCs, nor the ADC stage had a significant effect. Figure The CSF:plasma HIV decay ratios were variable . Exploration of the relationship of these ratios to baseline variables showed a high correlation with only the blood CD4 T lymphocyte count . Four were neurologically normal, while the fifth (subject 5007) had a diagnosis of ADC based on longstanding and clinically static myelopathy. The plasma and CSF HIV and WBC changes for these five subjects with The results of genotypic and phenotypic drug resistance analysis of plasma and CSF indicate that persistent plasma viremia was associated with drug resistance that was either demonstrable at baseline  or emerging during the period of observation (4015 and 4030). In two subjects (5001 and 5007) with baseline resistance, minor differences in genotypic mutations suggested at least partial compartmentalization, with CSF viruses more drug-susceptible than the predominant plasma quasispecies. Phenotypic drug susceptibility testing in Subject 5001 Figure  showed rThe other three  showed no evidence of compartmentalization at baseline, with similar susceptibility in both plasma and CSF virus populations. In Subject 4001 Figure , high-leIn the 24 treatment-group subjects without active ADC at baseline, QNPZ-4 scores were stable or showed small increases over the course of observation (not shown). The four subjects in the treatment group who presented with active ADC  showed both clinical improvement and distinct increases in this performance score in response to initiation or change of antiretroviral therapy. The clinical histories, antiretroviral therapy regimens, laboratory measurements, and QNPZ-4 scores of these subjects are presented in Figure The CSF found in the ventriculo-leptomeningeal space is separated from systemic sources of virus, immune defenses, and antiviral drugs, and is easily sampled by lumbar puncture. Thus, study of CSF HIV infection can serve a valuable role in understanding the dynamics and mechanisms of infection within an isolated tissue compartment. Further, CSF infection serves as both a model of and window into brain infection, providing important insight into viral neuropathogenesis. This CSF 'compartment' can be viewed as parallel to the brain compartment, sharing some of its barrier features. However, exchanges between CSF and blood may differ from those between brain and blood, and as a result, tissue responses may differ from CSF responses in important ways . More di10 cpm though this difference also varied considerably, ranging from -1.32 to 4.08 log10 cpm. Our results extend previous reports [The studies described here used complimentary cross-sectional and longitudinal approaches to show that CSF infection is a nearly universal facet of the ecology of systemic HIV infection. CSF HIV RNA concentrations correlated with those of plasma, but were characteristically lower. The median difference between plasma and CSF HIV VLs (Δplasma:CSF) was 1.30 log reports ,7,28-31 CSF WBC counts were commonly elevated in this series, confirming findings reported earlier in the epidemic . Our stcontribute to raising the CSF VL, or alternatively, represent only a response to high CSF virus. We discuss these two alternative hypotheses in relation to models of CSF infection below.While this combined experience indicates that CSF VLs and WBC counts are related, it begs the fundamental mechanistic question of whether the CSF WBCs actually Two of the observations reported here show a change in the relation of CSF infection to systemic infection as the latter progresses (as indicated by reduction in blood CD4 lymphocyte counts). First, in the cross-sectional study the Δplasma:CSF values in subjects with CD4 counts below 50 cells/μL (the first CD4 quartile of the group) were significantly larger than those of the remaining subjects with more preserved CD4 counts. This was related to higher plasma VLs, but without concomitant increase in CSF VLs; rather CSF VLs for these subjects were similar to those of the remaining subjects. This quartile also had lower CSF WBC counts. This observation confirms the impression of earlier investigators that CSF cell counts decrease in those with more advanced systemic infection . We discThe second difference was in the rates of viral decay during the initial phase of therapy in CSF compared to plasma. Viral decay in CSF was slower than in plasma in subjects with lower CD4 counts, while decay rates in the two fluids were more often equal at higher CD4 counts. This finding extends our earlier report of this association . It agre10 cpm in plasma and CSF, respectively) than in treated (median 4.00 and 1.66 log10 cpm in plasma and CSF) subjects (p < 0.001 for both variables compared by t-test). As noted also, HIV RNA was below the detection limit in a greater proportion of the CSF (38.9%) than plasma samples (18.5%). Given that the CSF HIV responded to therapy at least as effectively as plasma HIV, a relatively impaired CSF penetration of antiretroviral drugs seems unlikely to have influenced our findings. However, we did compare the CSF penetration of the antiretroviral regimens between those 'failing' to achieve virological suppression in plasma, and those with viral suppression, using a summed score in which each drug known to penetrate CSF counted as one and those not penetrating well counted as zero (data not shown). The means of the summed scores in the two groups  were not significantly different (p = 0.115). Of note, high CSF HIV was noted in rare treated subjects, sometimes exceeding plasma levels, as in subject 4034 who was treated with an unusual, poorly CSF-penetrating drug regimen [on therapy, he likely was on intermittent and ineffective ART at best [Before undertaking this study, we were concerned that CSF infection might not respond as well as systemic infection to treatment because of restricted penetration of many antiretroviral drugs . This wa regimen  but resp at best . OverallThe effectiveness of ART was clearly evident in the longitudinal treatment series. While the initial rate of CSF HIV decay lagged behind that of plasma in some subjects, all those with long-term follow-up achieved gratifying HIV reduction in this compartment with a time course generally proportional to their baseline level Figure . None ofIn addition to the generally favorable effects of ART on CSF infection, we observed an intriguing, salutary facet of treatment response. Namely, CSF infection showed a proportionally greater response to treatment than plasma infection in the face of antiviral drug resistance. Since drug resistance is the principal reason for long-term treatment failure, this may have very important implications for the prevention and treatment of CNS HIV infection and ADC. This superior effect of ART on CSF infection was noted in both the cross-sectional study and in the longitudinal treatment study. Moreover, it had been anticipated in our previous observations of CSF rebound in subjects with virological failure who underwent STI .treatment failures) with the 46 untreated subjects. While these two groups had similar plasma VLs , their CSF HIV RNA levels differed substantially . As a result, the Δplasma:CSF was about ten-fold higher in the treatment failure  than in the untreated group ; this 100-fold difference between plasma and CSF was similar to that reported by Stingele and colleagues in a study of paired specimens evaluating resistance mutations ['treatment failure' in the cross-sectional group was often associated with proportionally greater VL reductions in CSF than plasma.To examine this issue in the cross-sectional group, we compared the 36 subjects who were on therapy but had plasma VLs above 500 cpm  with high CSF HIV RNA , and small Δplasma:CSF might relate to exuberant cell entry and a high level of transitory or amplified infection as diagrammed in Figure oformans ,50, whertransitory component of infection may be involved, either because of insufficient CD4 target cells or reduced signals and responses to cell activation. In the case of low CD4 cells, the reduced transitory component uncovers low-level autonomous CSF infection. Of course, comparison of this setting with ADC in which CD4 cells are often decreased underscores a switch from low-level and seemingly benign autonomous infection to more active and 'malignant' macrophage infection with resultant toxic sequelae. This then raises the question of what causes the change in the character of autonomous infection in this setting – a change in the virus population or an alteration in the host? The increased Δplasma:CSF in treatment failure is supported by observations of diminished cell activation in this setting [Figure  setting , but is  setting  or simplThese considerations also do not fully preclude the importance of intrathecal drug effects. Simply considering extracellular drug levels in CSF compared to plasma may not be sufficient. Intracellular drug activity of nucleoside RT inhibitors might be enhanced within the cells supporting autonomous CNS infection despite lower extracellular drug concentrations than in systemic sites of infection . Subjectaseptic meningitis and ADC. The first is a component of the central focus of the study, while the second stands as an important background issue.These studies have relevance for two clinical neurological disorders related to CNS HIV infection: Aseptic or HIV meningitis is the clinical diagnosis conferred on HIV-infected patients with CSF pleocytosis without alternative cause, irrespective of clinical symptoms or signs such as headache, photophobia and stiff neck [iff neck -56. We eThe frequency of pleocytosis in our subjects and their lack of symptoms confirms earlier observations reported at a time when HIV infection was less well characterized ,3. Our saseptic meningitis in this setting should be reserved for those with symptoms or signs accompanying CSF abnormalities. The larger group of individuals with clinically silent elevated CSF cell counts may simply be designated as having asymptomatic pleocytosis of HIV infection.Since CSF pleocytosis is frequent and characteristically asymptomatic, how does one approach the HIV-infected patient presenting with headache who has an elevated CSF lymphocyte count ? When isADC is considered to be caused by brain HIV infection, but mediated by 'indirect' mechanisms involving a variety of pathways initiated and sustained by viral and host gene products [products -61. Infeproducts . The curNeurological performance was assessed using the QNPZ-4 score. This correlated highly with the presence and stage of ADC; the reduced QNPZ-4 score (median -0.50) of the cross-sectional sample was largely the result of including the ADC subjects. Longitudinally, the QNPZ-4 score afforded a stable measure in those without change in clinical neurological status, while it tracked a pattern of improvement in ADC subjects responding to ART that mirrored their antiviral response in CSF and plasma Figure . Cross-sThis does not mean that HIV brain infection is not central to the pathogenesis of ADC or that HIV brain infection cannot be reflected in CSF, but only that such an association is obscured by other factors. In this study, there may have been two principal reasons why no association was found. First, there was a high 'background' of increased CSF HIV in neurologically normal subjects. Second, many of our ADC subjects were on ART and clinically stable, and therefore likely suffered 'inactive' or residual brain disease. The lack of diagnostic specificity of CSF HIV RNA measurements underscores the varied relationship between CSF changes, on the one hand, and brain infection and disease, on the other. The CSF space and brain parenchyma are best viewed as two separate but intersecting compartments in which infection is not always congruent, necessitating caution in interpreting brain events from CSF findings. At least three types of intersections may occur. First, brain infection can 'overflow' into the CSF so that brain-derived HIV (and other markers) can be detected and directly measured in this fluid. The likely major cells of origin for HIV in these cases of autonomous infection are perivascular macrophages and perhaps the parenchymal macrophages and microglia , with viMeasuring the VL in CSF is not enough to make these important distinctions, and the CSF HIV RNA concentration cannot, in isolation, serve as a reliable diagnostic marker for ADC. Nor can the presence of pleocytosis be used to rule in or out ADC. This is unfortunate since objective markers are needed both in the clinic for practical diagnosis and in the research setting to more precisely define therapeutic targets. In the occasional patient, such as those designated as outliers in Figure activity of ADC and underlying HIV encephalitis. While we included subjects in this study with treated ADC and suppressed CSF infection, this would not be appropriate in a clinical trial assessing ADC treatment. Measuring CSF HIV concentration may be helpful in this setting, since undetectable CSF HIV likely signals suppressed brain infection.An additional clinical need is for laboratory measures that assess the HIV infection of the CNS is a nearly ubiquitous facet of systemic infection, but varies in character and clinical consequences. From very early exposure during primary systemic infection, most HIV-infected patients experience chronic asymptomatic CNS infection. However, a few individuals will develop encephalitis presenting as ADC. CSF sampling provides a valuable window into this infection and its variability.transitory versus more autonomous infections with rapid versus slower turnover rates; (2) CSF lymphocytosis either causing or responding to local infection, and (3) infection of the meninges presenting as 'asymptomatic pleocytosis' versus more toxic parenchymal or perivascular infection leading to the brain dysfunction of ADC. These provide a framework for future studies examining the mechanisms of infection in molecular terms and with respect to cell and HIV exchange and compartmentalization.We have framed the discussion of our results in models of CSF infection. Embedded in these models are a number of dichotomies relating systemic to CNS infections: (1) On a more practical level, to the extent that CSF infection reflects infection of the brain, antiretroviral therapy is usually effective in suppressing CNS HIV replication. Our longitudinal observations show that CSF infection usually responds well to combination antiretroviral therapy, equaling or exceeding systemic responses as reflected in plasma. Even where resistance leads to virological failure and persistent plasma viremia, ART may have a salutary effect on CSF. While the mechanisms underlying these favorable treatment effects remain uncertain, these observations are consonant with other reports using less frequent monitoring and are therapeutically reassuring. Our findings suggest that favorable virological outcomes in the CSF are the rule rather than an exception. They are also consistent with clinical studies that report a falling incidence of ADC in the current treatment era .The author(s) declare that they have no competing interests.SS participated in analysis and interpretation of the data and helped to draft the manuscript. AN helped with subject evaluations. NL participated in analysis and preparation of the data. TL helped in the study planning and coordinated the viral load assays. CP participated in planning of the genotypic and phenotypic resistance studies. SD participated in the study planning and design. EP participated in planning and interpretation of the genotypic and phenotypic resistance studies, and helped prepare the manuscript. RWP conceived of the design and analysis of the study and drafted the manuscript.The pre-publication history for this paper can be accessed here:Table 2. Clinical data, ART regimen history, and genotypic analysis of resistance mutations from five subjects with dissociated treatment responses. Antiretroviral medications able to penetrate into the CSF  are indiClick here for file