Plasmodium falciparum malaria is endemic, viral encephalitis and cerebral malaria are found in the same population, and parasitemia with Plasmodium falciparum is common in asymptomatic children. The objective of this study was to investigate the cerebrospinal fluid (CSF) biochemistry in children with cerebral malaria compared to those with presumed viral encephalitis.In developing countries where We studied the following CSF parameters: cell count, glucose, protein, lactic dehydrogenase (LDH) and adenosine deaminase (ADA) levels, in children with cerebral malaria, with presumed viral encephalitis, and in control subjects who had a lumbar puncture after a febrile convulsion with postictal coma.p < 0.01). Patients with cerebral malaria had lower CSF white cell count, glucose, protein, LDH levels and CSF/serum ADA ratio compared to patients with presumed viral encephalitis. CSF/serum ADA ratio was lower in patients with cerebral malaria due to the fact that serum ADA levels were significantly higher in patients with cerebral malaria compared to the other two groups. A CSF/serum ADA ratio of <0.38 and a CSF glucose level of <3.4 mmol/l were selected as the cut-off values with the highest sensitivities and specificities for comparing the two conditions.We recruited 12 children with cerebral malaria, 14 children with presumed viral encephalitis and 20 controls prospectively, over 2 years in the Government General Hospital in Kakinada, India. Patients with cerebral malaria had significantly lower CSF glucose, and higher protein, LDH, CSF/blood LDH ratio and CSF ADA levels but a lower CSF/serum ADA ratio compared to controls (CSF/serum ADA ratio and CSF glucose levels were the best discriminators of cerebral malaria from presumed viral encephalitis in our study. Further studies are needed to explore their usefulness in epidemiological studies. Previous studies have compared cerebrospinal fluid (CSF) features of bacterial meningitis and cerebral malaria ,2. This P. falciparum blood stages and no other identified cause of an encephalopathy following the WHO definition [In a 2-year prospective study at Government General hospital in Kakinada, India we recruited with ethical approval and informed consent by parents or guardians, children with a clinical diagnosis of presumed viral encephalitis, cerebral malaria and controls. The definition of presumed viral encephalitis was a reduced level of consciousness and pyrexia that could not be explained by a metabolic abnormality, dehydration or shock, negative blood and CSF cultures, and a negative blood slide for malaria parasites with recovery without antibiotics or lack of response to broad-spectrum antibiotics . Cerebrafinition . We recrCSF was obtained by lumbar puncture as soon as possible after admission if there were no contraindications, and after informed consent by the parents. CSF cell count, glucose, protein and CSF and serum LDH levels were determined by standard methods as in a previous study published by this group . ADA levp-value < 0.05 was taken as indicator for a statistically significant difference. Statistical calculations were performed with SPSS version 13.0 and Epi Info 6.04b . Confidence intervals for parameters were calculated by Confidence Interval Analysis software.For multiple comparisons of data with an approximately normal distribution the one way analysis of variance (ANOVA) was used. Tukey's HSD test was used for post hoc analysis of these data. For non-parametric data the Kruskal-Wallis test was used. The statistical method used for univariate comparisons of continuous data was the Mann-Whitney test, and for categorical data the chi-square test. For determination of a cutoff value for a CSF parameter for possible discrimination between presumed viral encephalitis and cerebral malaria, the co-ordinate of a receiver operating characteristic (ROC) curve indicating the greatest sensitivity and specificity was chosen . The ROp > 0.05). Serum LDH levels were not significantly different between groups (p > 0.05). However, CSF/Serum LDH ratios for both patients with cerebral malaria and presumed viral encephalitis were significantly higher than controls.46 children were recruited over a 2-year period. Twelve patients had cerebral malaria, 14 patients presumed viral encephalitis and 20 patients were controls. Demographic and laboratory parameters including LDH and ADA CSF/serum ratios are listed in Table For markers with a significant difference between cerebral malaria and the presumed viral encephalitis groups, we determined cut-off values of CSF parameters with maximum sensitivities and specificities and positive and negative predictive values. The best cut-off values taken from the co-ordinates of the ROC curve (see methods section) below which cerebral malaria was more likely than presumed viral encephalitis were: CSF white cell count, 4 cells/μl; CSF protein level, 0.43 g/l; CSF glucose, 3.4 mmol/l; CSF LDH, 22.5 IU/ml; CSF/serum ADA ratio, 0.385  being above 90% if the values of these parameters were above this cut-off. Specificities for the parameters were 100% for CSF glucose and 100% for CSF/serum ADA ratio and better than for other parameters, i.e. presumed viral encephalitis was always present in any patient with a value above the cut-off. This resulted in a probability  of 100% of having cerebral malaria in children with parameters below this cut-off Table .Plasmodium falciparum is common in regions hyperendemic for this parasite. In many of these areas, particularly in South East Asia, viral encephalitis is also a public health problem. It is therefore important to investigate discriminating CSF features between encephalitis and cerebral malaria. Our study is the most detailed study on CSF parameters in cerebral malaria reported so far. We could not, however, exclude the possibility that patients labeled as having cerebral malaria had a combination of encephalitis and parasitaemia with P. falciparum, but the lack of white cells in their cerebrospinal fluid make this possibility seem very unlikely.This is to our knowledge the first study to investigate differences in CSF parameters between presumed viral encephalitis and cerebral malaria. Asymptomatic parasitaemia with A CSF glucose level below 3.4 mmol/l was the best discriminator of cerebral malaria from presumed viral encephalitis. This was partly due to the fact that in cerebral malaria CSF glucose levels were below normal range. Low CSF glucose is a well-known phenomenon in cerebral malaria and CSF glucose has been found to be lower in patients with fatal cerebral malaria as compared to survivors . HoweverPrevious studies with data on CSF in cerebral malaria also found increased total protein levels ,18. Our P. falciparum malaria but serum ADA levels were found to be elevated to more than twice the level of controls in a previous study on patients with Plasmodium vivax malaria [We found that a CSF/serum ADA ratio of <0.38 was the best discriminator of cerebral malaria from presumed viral encephalitis. CSF ADA levels were measured in 3 patients with cerebral malaria previously, and found to have a mean (SD) of 6.6 (1.03) IU/l , slightl malaria . CSF ADA malaria .Mean CSF LDH levels of 26.5 U/ml in our patients with encephalitis were similar to the means of 22.6 and 22.3 U/ml found in two previous studies, respectively, ,24. Our CSF lactate, which is characteristically elevated in cerebral malaria , is anotPatients with clinical features of encephalitis and a positive malaria blood slide should still be treated as suffering from cerebral malaria regardless of the CSF findings, as our study cannot exclude a possible overlap in CSF/serum ADA ratios or CSF glucose levels between patients with encephalitis and cerebral malaria in other settings. The same applies to epidemiological surveillance pending further studies.P. falciparum and for example, Japanese encephalitis viruses, are both endemic are needed to validate our findings in different settings.CSF adenosine deaminase alone was not a useful discriminator between encephalitis and cerebral malaria. However, the CSF/serum ADA ratio was lower in patients with cerebral malaria due to the high levels of ADA in peripheral blood. This ratio should be evaluated in future, larger-scale, investigations into discriminatory factors between encephalitis and cerebral malaria. Although CSF glucose was significantly reduced in malaria patients, the results should be interpreted cautiously and require confirmation in a larger study. It would be preferable to use CSF/serum glucose ratios for future studies. Large-scale prospective studies in areas where The author(s) declare that they have no competing interests.SRJ participated in the design of the study, recruitment of patients, collection of samples and data-analysis. SV participated in the laboratory analysis and data analysis. RMA participated in design of the study. ME participated in data-analysis, interpreted the data and wrote the paper.


The central nervous system (CNS) is extremely vulnerable to ischemic injury. The details underlying this susceptibility are not completely understood. Since the CNS is surrounded by cerebrospinal fluid (CSF) that contains a low concentration of plasma protein, we examined the effect of changing the CSF in the evolution of CNS injury during ischemic insult.Lumbar spinal cord ischemia was induced in rabbits by cross-clamping the descending abdominal aorta for 1 h, 2 h or 3 h followed by 7 d of reperfusion. Prior to ischemia, rabbits were subjected to the following procedures; 1) CSF depletion, 2) CSF replenishment at 0 mmHg intracranial pressure (ICP), and 3) replacement of CSF with 8% albumin- or 1% gelatin-modified artificial CSF, respectively. Motor function of the hind limbs and histopathological changes of the spinal cord were scored. Post-ischemic microcirculation of the spinal cord was visualized by fluorescein isothiocyanate (FITC) albumin.The severity of histopathological damage paralleled the neurological deficit scores. Paraplegia and associated histopathological changes were accompanied by a clear post-ischemic deficit in blood perfusion.Spinal cord ischemia for 1 h resulted in permanent paraplegia in the control group. Depletion of the CSF significantly prevented paraplegia. CSF replenishment with the ICP reduced to 0 mmHg, did not prevent paraplegia. Replacement of CSF with albumin- or gelatin-modified artificial CSF prevented paraplegia in rabbits even when the ICP was maintained at 10–15 mmHg.We conclude that the presence of normal CSF may contribute to the vulnerability of the spinal cord to ischemic injury. Depletion of the CSF or replacement of the CSF with an albumin- or gelatin-modified artificial CSF can be neuroprotective. The central nervous system (CNS) including brain and spinal cord is extremely susceptible to hypoxic-ischemic insults compared with peripheral organ systems such as the liver, kidney, lung, or intestines. The mechanism underlying this susceptibility is not completely understood. Many theories have been proposed and intensively investigated, including the involvement of oxygen free radicals, calcium overloading, excitatory amino acid release and nitric oxide -3. TheseIn peripheral organ systems, capillaries are relatively permeable to proteins. The associated lymphatic system maintains lymph and interstitial fluid (ISF) protein concentration at about 2 g/dl, and ensures a negative interstitial pressure at about -3 mmHg . In the The spinal cord has a linear contour, it is therefore possible to create a CSF free environment for the lumbar spinal cord by removing the CSF and adjusting body position. Using a rabbit model of spinal cord ischemia, this investigation distinguished the influence of the CSF from that of ICP, and demonstrated that the addition of albumin or gelatin to the CSF can prevent injury and may be neuroprotective.Male New Zealand white rabbits, n = 77, weighing 2.5–3 kg were used . Animal studies were approved by the institutional animal care and use committee (permission number 2249) and complied with the 'Principles of Laboratory Animal Care' .Each rabbit was anesthetized with isoflurane in 100% oxygen  by facemask. Body temperature was maintained at 37 ± 1°C with a heating blanket. The right femoral artery was catheterized for monitoring mean arterial pressure (MAP) and for acquiring samples for blood gas analysis during surgical procedures. To remove the CSF and to monitor the ICP, a small silicon tube  was placed in the cisterna magna. Briefly, an incision was made on the dorsal neck to expose the nuchal ridge and dorsal cervical musculature. The atlanto-occipital membrane was exposed by blunt dissection and pierced with a 30 gauge needle. The silicon tube was twisted gently through the hole on the membrane until CSF was noted to pulsate in the catheter. The tube was fixed in position and immobilized to the adjacent muscle by an instant adhesive cyanoacrylate gel . To facilitate removal of the CSF and to administer albumin- and gelatin-modified artificial CSF another silicon tube was placed in the lumbar subarachnoid space. Briefly, a laminectomy was carried out at sacrococcygeal level to expose the lumbar subarachnoid space. The dura was then pierced with a 30 gauge needle. The tube was twisted gently into subarachnoid space through the dura and immobilized with the same adhesive to the adjacent muscle.Rabbits were randomly divided into 7 groups.No treatment was given. The ICP was monitored through the small silicon tube in the cisterna magna and maintained at 10 – 15 mmHg by withdrawing or infusing CSF through the tube in lumbar subarachnoid space. Rabbits were subjected to 1 h spinal cord ischemia.edures were used. First, while the tube in the cisterna magna was withdrawing CSF, the tube in lumbar subarachnoid space was kept open. Second, after removing the CSF, rabbits were kept in a tilted position with the head down (10 degrees) to prevent any remaining CSF from flowing to the lumbar region during the period of ischemia. After CSF depletion, rabbits were subjected to 1 h spinal cord ischemia. The ICP was monitored and maintained at 0 mmHg during the period of ischemia.The objective was to provide the ischemic spinal cord with a relatively CSF free environment. Prior to spinal cord ischemia, the CSF was removed as completely as possible; usually 0.8 – 1.2 ml CSF could be withdrawn. To assist in maintaining a CSF free environment below the lumbar spinal cord, two additional procpost mortem from a rabbit weighing 2.5 kg. Second, rabbits were kept in a tilted position with head up (10 degrees) during the period of ischemia to allow any remaining CSF inside cranium to flow into the ischemic region of the spinal cord. After CSF replenishment, the rabbits were subjected to 1 h spinal cord ischemia. The ICP was monitored and maintained at 0 mmHg.The objective was to provide the ischemic spinal cord with a normal CSF environment while maintaining ICP at 0 mmHg. Prior to spinal cord ischemia, the CSF was removed as completely as possible as described above. To ensure that CSF was present in the lumbar spinal cord ischemic region, two procedures were used. First, 0.1 ml CSF was immediately replenished through the tube in the lumbar subarachnoid space. This volume was previously determined by withdrawing CSF + 150 mEq/L, K+ 3.0 mEq/L, Mg++ 0.9 mEq/L, Ca++ 1.4 mEq/L, P 1.0 mEq/L, Cl- 155 mEq/L, glucose 60 mg/dl). Prior to spinal cord ischemia, 2 ml of albumn-modified artificial CSF was quickly flushed in through the silicon tube in the lumbar subarachnoid space and allowed to flow out through the tube in the cisterna magna. Rabbits were then subjected to 1 h spinal cord ischemia, during which time albumin-modified artificial CSF was continuously infused at rate of 2 ml/h. The ICP was monitored and maintained at 10–15 mmHg during the infusion by withdrawing or infusing the albumin-modified artificial CSF from the lumbar subarachnoid space.The objective of this group was to replace the CSF with albumin-modified artificial CSF in the spinal subarachnoid space. Albumin-modified artificial CSF was made by dissolving 8% bovine albumin  in artificial CSF  in artificial CSF. The experiment was performed as described for group 4.Treatment was the same as group 2, except that all rabbits were subjected to 2 h spinal cord ischemia and the CSF removal procedure was repeated every h.Treatment was the same as group 2, except that all rabbits were subjected to 3 h spinal cord ischemia and the CSF removal procedure was repeated every h.After the treatment according experimental design, spinal cord ischemia was induced according to Zivin . Under aAfter ischemic injury, each rabbit was tested daily for seven days. Motor function of the hind limbs was scored from 0 – 5 according to modified Tarlov's score  by an inAfter seven days of neurological evaluation, rabbits were euthanized with an overdose of pentobarbital sodium (200 mg/kg) injection via the heart. The spinal cord was perfused with 10% buffered formalin. The spinal cords from lower thoracic level to the lower lumbar level were harvested by multiple laminectomies and fixed in a 10% buffered formalin solution for one week. Spinal cord segments at the lumbar enlargement were embedded in paraffin. Transverse sections were cut at 6 μm and stained with hematoxylin-eosin. The spinal cord lesions at L5 level were scored under the microscope at a magnification of 40× and 100× by an investigator blinded to experimental procedures according to the following criteria -11:Score 1: Number of vacuolations < 5 in grey matter of the hemi-section.Score 2: Number of vacuolations ≥ 5 in grey matter of the hemi-section.Score 3: The architecture of grey matter was necrotic or completely destroyed or replaced by scar tissue. No neurons could be seen.At 23 h after spinal cord ischemia, lumbar spinal cord microcirculation was studied in two rabbits from groups 1–5. Each rabbit received 0.3 g/kg 15% fluorescein isothiocyante (FITC) albumin (Sigma-Aldrich) by i.v. bolus injection . Rabbitsp value < 0.05 was considered statistically significant.The physiological parameters were analyzed with one-way repeated measures ANOVA  followed by Tukey test. The neurological deficit scores and the histopathological scores were analyzed with Kruskal-Wallis ANOVA on Ranks followed by the Tukey test. A 2, PCO2, pH and glucose in any group . The CSF depletion with the ICP maintained at 0 mmHg prevented the development of paraplegia . However, replenishment of CSF around ischemic spinal cord  did not prevent paraplegia, even when the ICP was controlled at 0 mmHg . Replacing the CSF with albumin- or gelatin-modified artificial CSF under physiological ICP conditions (10–15 mmHg) significantly prevented the development of paraplegia .The neurological deficit score, taken 24 h after ischemia did not differ from the score obtained seven days after ischemia. Control animals subjected to 1 h of spinal cord ischemia demonstrated permanent paraplegia , but did not significantly prevent paraplegia after 3 h of ischemia .Similarly, the CSF depletion significantly prevented paraplegia in rabbits subjected to 2 h of spinal cord ischemia . After ischemia the 'no-reflow' phenomenon was characterized by the absence of capillary filling in both the grey matter and white matter. A 'no-reflow' phenomenon of lumbar spinal cord was demonstrated in control rabbits Fig . In contThe CNS lacks a lymphatic system; instead it is surrounded by the CSF. The CSF is very different from the lymph in peripheral tissues in at least two aspects: protein concentration and the resultant interstitial fluid pressure.In peripheral tissues, capillaries are relatively permeable and as a result the ISF contains about 2 g/dl of plasma proteins. It is believed that interstitial proteins and hyaluronic acids form a dense network of proteoglycan filaments that impede fluid flow through the interstitium. Normally the amount of free-flowing fluid, present in the interstitium is small. A low interstitial protein concentration results in an increased amount of free ISF. An elevated concentration of interstitial protein may reduce the free ISF, but it also attracts more fluid, resulting in increased volume. The lymphatic system is the scavenging pathway for interstitial proteins. By regulating the removal of excess protein, the lymphatic system keeps the interstitial protein concentration around 2 g/dl. This ensures limited free fluid and also regulates the ISF volume. Lymph flow reduces ISF volume resulting in negative interstitial pressure. Therefore, the movement of proteins from plasma to ISF and finally to lymph is important for maintaining extracellular homeostasis.+ in the ISF have the potential to move easily into cells. More importantly, the CSF is contiguous with the ISF, with the Virchow-Robin spaces, serving as a conduit. To make matters worse, the ICP averages about 10 mmHg leading to a positive interstitial fluid pressure. Taken together, these factors make the CNS prone to edema formation. As a result cells in the CNS constantly consume energy to remove excess intracellular fluid in physiological condition. When cell energy is compromised, such as in ischemia, or when the cell membrane is damaged by direct trauma, cells rapidly become swollen, i.e. cytotoxic edema . Depletion of the CSF creates a partially CSF free environment in the ischemic spinal cord region. Although depletion of the CSF does not eliminate ISF, it limits the extent to which the ISF can be replenished from the CSF and limits occurrence of edema. It is estimated that the total volume of CSF is about 2.3 ml or 0.67 ml/kg for an adult rabbit, and the CSF production rate is reportedly to be between 7.8–8.8 μl/minute -18. We wUsing albumin-modified artificial CSF to replace the CSF significantly prevented paraplegia even when the ICP was maintained within the physiological range. These data indicate that detrimental effect of the CSF may be largely ascribed to its low protein concentration. A high concentration of protein present in artificial CSF not only provides limited free fluid to ISF, but may also increase the protein concentration in ISF resulting in reduced amount of the free fluid in interstitium. The increase of protein concentration in ISF was less likely to result in volume expansion in the interstitium because of the protein concentration gradient between the CSF and the ISF; any ISF volume increase would be transferred to the protein-modified artificial CSF. We believe that the ultimate reason why protein is important for the extracellular environment may be due chiefly to its water and ion binding capacity. The water binding capacity for albumin is so large that it is estimated that one gram of albumin can bind 18 ml of water ,20. A nuRabbits with albumin- or gelatin-modified artificial CSF replacement maintained with a physiological ICP still showed ~20% paraplegic rate, while depletion of the CSF showed 0% paraplegic rate, although the difference was not statistically significant by Fisher exact test. The effect of depleting the CSF may be partially due to ICP reduction, and the 20% paraplegic rate may be explained by the presence of physiological ICP. Elevated ICP has been known to be clinically detrimental. For example, CSF drainage has been used for almost 50 years to prevent paraplegia during aortic surgery when the aorta needs to be cross-clamped ,29. WhilOur experiments demonstrated a clear 'no-reflow' and 'low-reflow' phenomenon, which was accompanied by paraplegia and histopathological damage in the spinal cord. Depletion of the CSF or replacement with albumin- or gelatin-modified artificial CSF prevented the 'no-reflow' phenomenon, associated motor function deficit and histopathological spinal cord damage. Although many investigators reported blood perfusion deficits following brain and spinal cord injuries -44, the It has been shown that just 25 minutes of spinal cord ischemia in this rabbit model is enough to cause permanent paraplegia ,15. In oIn summary, our findings suggest that the extracellular environment, consisting of a low protein concentration in the CSF and the ISF and a positive interstitial pressure, may determine the vulnerability of CNS to ischemia. This study has determined that depletion of the CSF or replacing the CSF with albumin-, or gelatin-modified artificial CSF in combination with a lowered ICP, provides protection in the setting of ischemic injury to the CNS.The author(s) declare that they have no competing interests.YFW: conceived of the study, designed and coordinated all experiments, participated in spinal cord ischemia experiment in rabbits and drafted the manuscript.JKG: helped in the preparation of the manuscript and data analysis.GZ: participated in spinal cord ischemia experiment in rabbits.SGS: participated in data analysis and helped in the revision of the manuscript.SH: participated in spinal cord ischemia experiment in rabbits.YW: participated in spinal cord ischemia experiment in rabbits.All authors have read and approved the final manuscript.


Two retrovirus vectors were compared for their ability to express granulocyte-macrophage colony stimulating factor (GM-CSF) in a haematopoietic cell line, FDCP1, which is dependent on GM-CSF for survival. Both a MoMLV-based vector pVneoGM, and a MPSV-based vector, M3neoGM, were found to be capable of transmitting and expressing both GM-CSF and neomycin sequences in the myeloid FDCP1 cell line. Our results also demonstrate that pVneoGM is more efficient at generating GM-CSF independent colonies than M3neoGM. Analysis of cell lines derived after infection confirmed pVneoGM expressed higher levels of GM-CSF. Cell lines generated by infection with pVneoGM responded to levels of exogenous recombinant GM-CSF which did not stimulate growth of the parental cell line, suggesting autocrine stimulation may convey a proliferative advantage under sub-optimal growth conditions. Finally the parental vectors pVneo and M3neo were shown to be capable of expressing the neomycin gene in both murine haematopoietic progenitor and stem cells.


Leukocytosis sometimes accompanies malignant neoplasms in the absence of infection. It is thought that the production of colony-stimulating factor by neoplasms is the most potent cause of tumour-induced leukocytosis; several mechanisms have been suggested to explain this. We examined 155 human tumour xenografts established in nude mice, and found that 17 of the xenografts induced remarkable leukocytosis  in nude rats. We examined granulocyte colony-stimulating factor (G-CSF) production by the xenografts to study the mechanisms underlying this tumour-induced leukocytosis. Ten of the 17 xenografted human tumours appeared to express the G-CSF gene. Serum G-CSF increased, to concentrations of 179-37,218 pg ml-1, in host animals transplanted with the ten xenografts expressing the G-CSF gene transcripts. The biological activity of serum G-CSF also increased, to concentrations of 206-9,074 pg ml-1, in the host animals transplanted with the ten xenografts. Immunohistochemical analysis demonstrated G-CSF production at the cellular level in three of the ten xenografts. These results suggested that the production of G-CSF is a common event in human tumour xenografts associated with leukocytosis, but that factors other than G-CSF are also likely to be involved. Leukocytosis induced by neoplasms seems to be a heterogeneous and complex disorder.


The gut hormone, ghrelin, is involved in the neuroendocrine and metabolic responses to hunger. In monogastric species, circulating ghrelin levels show clear meal-related and body weight-related changes. The pattern of secretion and its role in ruminant species is less clear. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation. There is also evidence that ghrelin is involved in reproductive function. In the present study we used immunohistochemistry to investigate the presence of ghrelin and GHSR-1a in sheep reproductive tissues. In addition, we examined whether ghrelin and GHSR-1a protein expression is developmentally regulated in the adult and fetal ovine testis, and whether there is an association with markers of cellular proliferation, i.e. stem cell factor (SCF) and proliferating cell nuclear antigen (PCNA).Antibodies raised against ghrelin and its functional receptor, GHSR-type 1a, were used in standard immunohistochemical protocols on various reproductive tissues collected from adult and fetal sheep. GHSR-1a mRNA presence was also confirmed by in situ hybridisation. SCF and PCNA immunoexpression was investigated in fetal testicular samples. Adult and fetal testicular immunostaining for ghrelin, GHSR-1a, SCF and PCNA was analysed using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fisher's least significant difference.In adult sheep tissue, ghrelin and GHSR-1a immunostaining was detected in the stomach (abomasum), anterior pituitary gland, testis, ovary, and hypothalamic and hindbrain regions of the brain. In the adult testis, there was a significant effect of season (photoperiod) on the level of immunostaining for ghrelin (p < 0.01) and GHSR-1a (p < 0.05). In the fetal sheep testis, there was a significant effect of gestational age on the level of immunostaining for ghrelin (p < 0.001), GHSR-1a (p < 0.05), SCF (p < 0.05) and PCNA (p < 0.01).Evidence is presented for the presence of ghrelin and its receptor in various reproductive tissues of the adult and fetal sheep. In addition, the data indicate that testicular expression of ghrelin and its receptor is physiologically regulated in the adult and developmentally regulated in the fetus. Therefore, the ghrelin ligand/receptor system may have a role (endocrine and/or paracrine) in the development (cellular proliferation) and function of the reproductive axis of the sheep. Slides were treated with RNase A, desalted, with a final high stringency wash (30 min) in 0.1 × SSC at 60°C, dried and exposed for 4 weeks to Kodak Biomax MR Film . Autoradiographic images were captured using the Image-Pro Plus system .Frozen adult sheep testicular sections (20 μm) were cut onto glass slides double-coated with gelatin and poly-L-lysine, with six or seven sections mounted on each slide. Briefly, slides were fixed in cold NBF, acetylated, and hybridised overnight at 58°C using [Immunostained area for ghrelin and GHSR-1a in adult testis tissue from the Soay rams, and ghrelin, GHSR-1a, SCF and PCNA in fetal testis tissue was analysed using computer aided image analysis. The system was composed of a Zeiss axioplan microscope  and HV-C20 Hitachi camera  connected to a computer running Image-Pro Plus system. Four sections per testis were quantified for tissue section area stained (brown colour) over six randomly selected fields of view previously shown to be sufficient to stabilise the mean and standard error . The totImage analysis data were subjected to one-way ANOVA, with differences between testicular immunostaining levels of ghrelin, GHSR-1a, SCF and PCNA at different stages of gestation in the fetuses determined by Fisher's least significant difference.The specificity of the three antibodies  was confirmed by the absence of immunostaining when the primary antibody was replaced with serum from the species in which the antibody was raised. Additionally, immunostaining was abolished when the antisera were pre-incubated with the immunising peptide , arcuate nucleus (ARC), ventromedial hypothalamus (VMH) and ependymal lining (EL) of the third cerebral ventricle Fig. . GhrelinTestis size was reduced in the LD group (data not shown), as expected . GhrelinThe ovine fetal testis is visible as an outgrowth of undifferentiated cells from the mesonephros at gestational day 30 Fig. . At thisAt day 30, low level immunostaining for ghrelin was observed in the gonadal/mesonephros complex Fig. . From daCells stained for SCF were most prominent in the fetal testis at day 30 Fig. . The latThere was a significant overall effect of gestational age on the immunostaining levels of ghrelin in the fetal testis , in reproductive tissues of the sheep. Novel data are presented that indicate that ghrelin and its functional receptor, GHSR-1a, are regulated during development of the ovine fetal testis and by seasonal developmental changes in the adult testis. Moreover, the developmental pattern of expression corresponds with the postulate by Barreiro and colleagues  that theIn the sheep brain, ghrelin and GHSR-1a immunoreactivity were demonstrated in the hypothalamic region, including the median eminence (ME), arcuate nucleus (ARC), ventromedial hypothalamus (VMH) and ependymal lining (EL) of the third cerebral ventricle. The presence of ghrelin and GHSR-1a in the hypothalamus is consistent with its role in the regulation of food intake . Howeverin vitro [In the sheep anterior pituitary gland, ghrelin and GHSR-1a were ubiquitously expressed. Ghrelin gene expression has been found in the pituitary glands of rodents, pigs and humans ,21,58. Iin vitro . Particuin vitro  that theGhrelin and GHSR-1a immunostaining were detected in sheep ovarian follicles at all developmental stages, mainly in the granulosa cells. Caminos et al.  and Gaytin vitro, and to modulate Leydig cell proliferation in vivo and the expression of relevant testicular genes, such as that encoding stem cell factor (SCF) [In the adult sheep testis, strong ghrelin immunostaining was evident in the interstitial area where Leydig cells are localised. Staining was also present in the germ and Sertoli cells, with an indication of increased ghrelin immunoreactivity in the germ cells during the mitotic phases and meiotic pro-phases of the spermatogenic cycle. GHSR-1a protein was detected in the interstitial Leydig cell containing area of the testis, as well as in the Sertoli and germ cells within the tubules, and the pattern of GHSR-1a mRNA expression across the testis indicated that the mRNA was present in the interstitial area and around the periphery of the tubules. Ghrelin immunostaining has been demonstrated in interstitial Leydig cells and, at lower intensity, in Sertoli cells of the rat . Ghrelinor (SCF) . In the or (SCF) ,30. It hor (SCF) . Whetheror (SCF) .In the fetal sheep testis, the present findings indicate that the expression of ghrelin and GHSR-1a protein is linked to gestational age. Differentiation of the fetal gonad begins with the development of the gonadal ridges from thickening of the ventrolateral surface of the embryonic mesonephros. The genital ridge is composed of somatic cells from the mesonephros and migratory primordial germ cells originating from the extraembryonic mesoderm. In the fetal sheep, morphological sexual differentiation of the gonads begins around day 27 of gestation, and germ cell migration is complete just after day 30 ,71. In tThe present data indicate that both components (ligand and receptor) of the ghrelin signalling system are present in tissues of the reproductive axis of the sheep. The ligand and receptor are developmentally regulated in the fetal testis and physiologically regulated by photoperiod in the adult testis. These findings are consistent with both a central endocrine role and a potential peripheral paracrine/endocrine regulatory role for ghrelin in the control of reproductive tissue development and function in sheep. Further studies are needed to identify the precise functional role of the ghrelin system in the reproductive axis.in situ analyses of ghrelin and GHSR-1a, statistical analyses and drafting the manuscript. YAB participated in the immunological analysis of GHSR-1a, statistical analysis and drafting the manuscript. UD participated in the immunological analysis of SCF, statistical analysis and drafting the manuscript. AL participated in the immunological analysis of PCNA, statistical analysis and drafting the manuscript. RGL participated in the design of the studies, all immunological analyses, statistical analyses and drafting the manuscript. CLA participated in the design of the studies, collection of tissues, in situ analysis, statistical analyses and drafting the manuscript.DWM participated in the design of the studies, collection of tissues, all immunological analyses, statistical analyses and drafting the manuscript. JLH participated in the design of the studies, collection of tissues, immunological and


Pseudomonas aeruginosa O12, occurred. Our case showed permanent ventricular-to-lumbar CSF gradients of leukocytes, protein and glucose that were increased during the acute phase of meningitis, with the greatest amplitude being observed when bacteria were present in both ventricular and lumbar CSF. This might suggest a greater extent of meningeal inflammation in the lumbar than in the ventricular region. Our case also showed that the increase in intravenous antibiotics (cefepim from 8 to 12 g/day and ciprofloxacine from 1.2 to 2.4 g/day) led to an increase in concentration in plasma but not in CSF.Differences in the composition of ventricular and lumbar cerebrospinal fluid (CSF) based on single pairs of samples have previously been described. We describe a patient that developed post-surgical recurrent meningitis monitored by daily biochemical and bacteriological CSF analysis, simultaneously withdrawn from lumbar space and ventricles. A 20-year-old Caucasian man was admitted to the ICU after a resection of a chordoma that extended from the sphenoidal sinus to the anterior face of C2. CSF was continuously leaking into the pharyngeal cavity after surgery, and three episodes of recurrent meningitis, all due to  Diagnosis is based almost exclusively on biochemical and bacteriological analysis of cerebrospinal fluid (CSF) withdrawn either by puncture in the lumbar space or through an external drain located either in the lumbar or ventricular space. It is established that CSF infection is strongly suspected in the presence of a positive CSF culture and/or of a CSF : serum glucose ratio of less than 0.6 and/or of a CSF leukocyte count of more than 11/mmDifferences in the composition of ventricular and lumbar CSF, based on single pairs of CSF samples, were previously described -4. ThesePseudomonas aeruginosa O12. The last two episodes were monitored by daily biochemical and bacteriological analysis of CSF withdrawn in parallel from the lumbar space and ventricles by external lumbar drainage (ELD) and external ventricular drainage (EVD).Here we describe a patient who developed, after surgery for a chordoma of the clivus, three episodes of recurrent meningitis due to A 20-year-old Caucasian man with no medical history was admitted for elective surgery of a chordoma that extended from the sphenoidal sinus to the anterior face of C2. The first surgical step consisted of a subtotal removal of the tumour by a transfrontal approach. An EVD was inserted at day 1 (D1) because of the appearance of hydrocephalia.3. CSF withdrawn through the ventricular drain showed CSF leukocytes at 830/mm3, a CSF protein concentration of 0.99 g/l and a CSF glucose concentration of 3 mmol/l (for a glycaemia of 6 mmol/l). A Ps. aeruginosa O12 resistant to almost all antibiotics except ceftazidime and polymyxin B, similar to that repeatedly found in the oral cavity, grew in CSF culture. It was therefore decided to replace the ventricular drain with another in the controlateral hemisphere for two purposes: first, to withdraw CSF to reduce CSF leakage by the fistula, and second, to perform a biochemical and bacteriological analysis. Antibiotherapy was started with intravenous (i.v.) ceftazidime  combined with amikacin and polymyxin B both in the ventricles.At D10, the second approach consisted in a transoral resection of the tumour with a reconstruction of the pharyngeal wall with skin taken from the arm. However, the wall was not totally occlusive, with a continuous CSF leak into the pharyngeal cavity. Seven days later (D17), the patient developed meningitis with fever and a white blood cell count of 13,800/mmA clear improvement in the meningitis allowed us to perform the third and last approach (at D42): an occipito-cervical fixation procedure with EVD removed. Three days later (D45), the patient developed a new episode of hydrocephalia. It was therefore decided to introduce ELD rather than EVD.3, a CSF protein concentration of 1.88 g/l and a glucose concentration of 0.9 mmol/l (for a glycaemia of 6 mmol/l). A CSF culture found the same bacteria as in the first episode of meningitis. This second episode was considered to be related to the persistent pharyngeal fistula. The ELD was replaced with a new one and EVD was added because of the suspicion of an additional obstruction in the 4th ventricle related to post-surgical oedema. Meningitis was treated with an increasing dose of i.v. ciprofloxacin  and i.v. cefepim  and amikacine and polymyxin B colistine both administered directly into the ventricles.Twelve days later (D57), the patient developed a second episode of meningitis: fever, lumbar CSF leukocytes at 14,000/mmPs. aeruginosa O12 in CSF culture, increased CSF protein and decreased CSF glucose levels in both ELD and EVD. Antibiotics were kept constant and, despite negative cultures, ELD and EVD were replaced with new drains. Interestingly, since this last episode of meningitis, the pharyngeal fistula disappeared, which indicated the end of pharyngeal contamination of CSF. The patient improved rapidly and was discharged home at D108. No further episode of meningitis during the next 3 years, nor any toxic effect related to the high doses of antibiotics, was observed. It is noteworthy that repetitive cerebral computed tomography scans showed no empyema.The third episode of meningitis appeared at D66 with identification of the same Ps. aeruginosa O12 was present in the meningeal cavity.Figure Ps. aeruginosa O12 were 16 and 0.25 mg/ml, respectively.Our case also showed that the increase in the amount of antibiotics given did increase their concentration in plasma but not in CSF. Indeed, i.v. cefepim was increased from 8 to 12 g/day and i.v. ciprofloxacin from 1.2 to 2.4 g/day from D64 to D95. This induced a persistent increase in plasma cefepim concentration from 46 μ g/ml to more than 60 μg/ml and plasma ciprofloxacin concentration from 0.2 μg/ml to more than 1.0 μg/ml. However, only a transient increase in cefepim concentration  and no increase in ciprofloxacin concentration (0.4–0.5 μg/ml from D63 to 95) were seen in lumbar and ventricular CSF. It is noteworthy that the inhibitory minimal concentrations of cefepim and ciprofloxacin for Ps. aeruginosa O12. It showed the presence of a rostrocaudal gradient of leukocytes and protein and an inverse gradient of glucose. This confirmed previous work that showed greater leukocytes and protein concentration in lumbar than in ventricular CSF in patients with a central neural system infection, mostly after neurosurgery [Our case report followed ventriculo-lumbar CSF gradients in leukocytes, protein and glucose concentration during two episodes of post-operative recurrent meningitis due to osurgery . Howeverosurgery .Ps. aeroginusa O12, in similar quantities, seemed to induce a greater alteration of meningeal permeability with greater leukocyte and protein concentrations and a lower glucose concentration in the lumbar than the ventricular CSF region. Although still debatable, the decrease in glucose concentration in CSF seems to be less related to a 'leukocyte-induced glucose consumption' but rather to a meningeal shift of glucose metabolism to anaerobic glycolysis, as indicated by the concomitant increase in CSF lactate concentration and/or a decrease in meningeal glucose transport [We extend previous studies by showing that the greatest amplitude of ventricular-to-lumbar gradients for all measured parameters  were seen during the very acute phase of meningitis, when bacteria were present in the meningeal cavity. The mechanisms of such ventricular-to-lumbar gradients are unknown. Our data strongly suggest a compartmentalization of meningeal inflammation in the ventricular and lumbar area. Indeed, similar bacteria, here ransport ; the latRecurrent meningitis led us to increase the antibiotic dosage to achieve a better concentration in CSF . SurprisIn summary, this case report shows that the maximal rostrocaudal gradient of leukocytes, protein and glucose was seen in the very acute phase of meningitis. This strongly suggests a greater alteration in the meningeal barrier and very probably a greater meningeal inflammation in the lumbar than the ventricular regions.Ps. aeruginosa O12.• 	The paper describes a patient that developed, after surgery for a chordoma of the clivus, three episodes of recurrent meningitis due to • 	Episodes were monitored by biochemical and bacteriological daily analysis of CSF withdrawn in parallel from lumbar space and ventricles by external lumbar and ventricular damamge.• 	We observed a permanent ventricular-to-lumbar CSF gradients of leukocytes, protein and glucose that increased during the acute phase of meningitis, with the greatest amplitude observed when bacteria was present in both ventricular and lumbar CSF.• 	This may suggest a greater extent of meningeal inflammation in lumbar than in ventricular regionCSF = cerebrospinal fluid; D1, day of insertion of EVD; ELD = external lumbar drainage; EVD = external ventricular drainage; ICU = intensive care unit; i.v. = intravenous.The author(s) declare that they have no competing interests.WN and AM coordinated the data analysis and drafted the manuscript. JM, LR and J-F T participated in bacteriological analysis. A-C L and BG participated in analysis of clinical data. DP helped to draft the manuscript. All authors read and approved the final manuscript


The neurofilament is a major structural component of myelinated axons. Increased cerebrospinal fluid (CSF) concentrations of the light chain of the neurofilament protein (NFL) can serve as a sensitive indicator of central nervous system (CNS) injury. To assess whether interrupting antiretroviral treatment of HIV infection might have a deleterious effect on the CNS, we measured NFL levels in HIV-infected subjects interrupting therapy.We identified subjects who had CSF HIV RNA concentrations below 50 copies/mL at the time combination antiretroviral therapy was interrupted, and for whom CSF samples were available before and after the interruption.A total of 8 subjects were studied. The median (range) CSF NFL level at baseline was <125 (<125–220) ng/L . All 8 subjects exhibited an increase in CSF and plasma HIV RNA after stopping therapy, accompanied by intrathecal immunoactivation as evidenced by CSF lymphocytic pleocytosis (7/8 patients) and increased CSF neopterin concentration (5/6 patients). Three subjects showed a consistent increase in CSF NFL, rising from <125 ng/L to a maximum of 880 (at day 148), 1,010 (day 58) and 10,930 ng/L (day 101). None exhibited new neurological symptoms or signs, or experienced functional deterioration during the period off treatment; of 5 who underwent brief quantitative neurological testing, none showed worsening performance.These findings suggest that resurgence of active HIV replication may result in measurable, albeit subclinical, CNS injury. Further studies are needed to define the frequency and pathobiological importance of the increase in CSF NFL. The mortality and morbidity of HIV infection have substantially decreased in the developed world over the past decade, largely due to the introduction of combination antiretroviral therapy (ART) . WidesprAlthough effective in suppressing infection, current ART regimens do not eradicate HIV, and prolonged treatment may be complicated by development of drug resistance and an array of side effects . A numbeWe undertook the current study to determine whether interrupting therapy can cause deleterious CNS effects that are not appreciated by clinical history and examination. We used archived specimens from study subjects who had interrupted therapy and been followed by repeated lumbar puncture (LP) and CSF analysis before and at various times after remaining off treatment. As a measure of neurological injury, we used concentrations of the light chain of neurofilament protein (NFL). NFL is a major structural component of myelinated axons, and the CSF level can serve as a sensitive marker of axonal damage in a number of conditions ,6. Both In a previous study, increased CSF NFL concentrations were found in 12 of 18 AIDS patients without CNS opportunistic infections, but in only three out of 12 subjects with less advanced, asymptomatic HIV-1 infection .We retrospectively identified subjects from two centers who underwent serial lumbar punctures before and after interruption of a combination antiretroviral regimen, who had undetectable HIV RNA levels in CSF at the time of the interruption and who had longitudinal samples available for our studies. All subjects participated in separate local protocols evaluating the CSF responses to changes in antiretroviral treatment. Decisions to interrupt treatment were made by the subjects and their primary caregivers independent of the CSF studies. The protocols were approved by University of California San Francisco (UCSF) Committee on Human Research (CHR) and by the Research Ethics Committee of Göteborg University. All subjects provided informed consent.CSF viral dynamics from some of these subjects have been previously described ,17.CSF concentrations of NFL were analysed using a previously described ELISA . In brieCell-free CSF and plasma HIV-1 RNA were quantified with the Amplicor HIV-1 Monitor assay, version 1.5  with a dynamic range down to 50 copies/mL and a detection limit of approximately 20 copies/mL.Neopterin was measured by a commercially available radio-immunoassay  with a normal reference value of ≤ 4.3 nmol/L in CSF .Routine CSF assessments included CSF white blood cell (WBC) count and peripheral blood CD4+ T-lymphocyte (CD4) cell determination. Albumin was measured using standard Clinical Laboratory methods in each center, and the CSF:serum albumin ratios were calculated [CSF albumin (mg/L)/serum albumin (g/L)] and used as a measure of blood-brain barrier integrity . The fivUnless otherwise indicated, descriptive group statistics in this small study are presented as median values with the range of values, and comparisons between subjects with and without increases in CSF NFL used the Mann-Whitney U test.Eight subjects, five from San Francisco, California, USA and three from Göteborg, Sweden were included in the study. Demographic and baseline characteristics of the subjects are listed in Table The time course of changes in subjects' salient laboratory findings are shown in Figure The peripheral blood CD4-cell count decreased from a median of 446 cells/μL  while on treatment to 258 (208–738) cells/μL during the off-treatment follow-up.10) copies/mL in all subjects, and the plasma HIV RNA levels was undetectable in six subjects. The median viral load increased to a maximum of 4.36 (3.38–4.87) log10 RNA copies/mL in CSF and to 5.23 (4.78–6.35) log10 copies/mL in plasma during the interruption period. All but one of the subjects developed an increase in CSF WBCs. CSF cell counts increased from a median of 0.5  cells/μL on treatment to a maximum of 14 (2–62) cells/μL after stopping therapy.All 8 subjects developed an increase in CSF and plasma HIV RNA concentrations. At baseline, the CSF HIV-1 RNA was <50 , while the remaining five subjects did not (panels D-H). As shown in the middle panels of each triplet set, all eight subjects had normal CSF NFL concentrations (<250 ng/L) while on treatment, with six of these below the detection limit (125 ng/L). For two subjects , CSF samples were also available before initiation of antiretroviral treatment, 9–14 months prior to baseline, and CSF NFL concentrations were found to be <125 ng/L at these intervals as well. Treatment interruption resulted in CSF NFL increases in the three subjects , rising from baselines at the detection limits to maximum values of 880 ng/L at day 148 (6011), 10,930 ng/L at day 101 (6012) and 1,010 ng/L at day 58 (223). These increases were delayed in relation to virological changes. In subject 223 the only interval testing after treatment stopped was at day 58, while in the two others who had more frequent measurements the earliest documented increase in NFL for subject 6012 was at day 59 and for subject 6011 the inflection point showing the initial increase was delayed to 86 and 99 days.In the two subjects with increases in NFL and more frequent measurements, the rise in plasma and CSF HIV RNA preceded that of NFL, with the plasma HIV rising by the first sampling at 17 and 15 days and the CSF by days 38 and 36. By contrast, the first change in NFL was delayed by an additional three to seven weeks. The change from baseline to maximum post-STI HIV RNA in blood and CSF was not notably different in the three subjects with increases in NFL compared to those without any change in this neural marker. There was no clear association of NFL elevation with that of CSF WBCs; indeed, the one subject without CSF pleocytosis  had the greatest increase in CSF NFL.The subjects were all clinically stable during the period of observation . None developed either AIDS-related events or neurological symptoms during the off-treatment follow-up. Likewise, their neurological examinations were unchanged, and in the five tested for quantitative neurological performance (including two with NFL elevations – subjects 6012 and 6011), none showed deterioration in their QNPZ-4 scores (shown in middle graphs of subjects' triplet panels).In keeping with the inflammatory response in CSF, all subjects in which CSF and serum neopterin were measured exhibited an increase in this marker while off therapy with the exception of subject 160  showed evidence of blood-brain barrier disruption with an elevated CSF:serum albumin ratio. In all others the albumin ratios were stable or showed only minor change that did not parallel changes in CSF NFL (data not shown). As expected within this small dataset, no statistical significant difference could be found between subjects with or without CSF NFL increase, neither regarding the magnitude of increase in CSF neopterin, CSF WBC, CSF nor plasma viral load (data not shown).While the use of STI to stimulate and boost the immune system by re-exposure to viral antigens has not been proved effective and may be complicated by enduring T cell loss and enhanced resistance ,21, therThe neurofilament is a major structural element of neurons, found most conspicuously in larger neurons and their myelinated axons. It is composed of a triplet protein of which the light subunit (NFL) is an essential component of the neurofilament core. Its main function is to maintain the axonal calibre and it thereby has a crucial role in the structural and functional integrity of axons and in their capacity to rapidly conduct nerve impulses . IncreasCSF NFL increased substantially in three of our eight subjects studied after stopping antiretroviral therapy, thereby indicating brain injury in this setting. The mechanisms responsible for this rise in NFL remain to be determined. Indeed, from a virological perspective, it is uncertain whether the increase in NFL is simply a complication of established viremia or is related more particularly to the abrupt surge in viral replication that follows treatment interruption. In other words, was this neurological damage due simply to higher levels of HIV replication or due to the "shock" associated with a rapid resurgence after interruption? Perhaps favouring the latter is the finding of elevations in NFL in these subjects without an AIDS diagnosis and CD4 counts above 200 cells/μL, contrasting somewhat with our earlier study that showing CSF NFL elevations chiefly in subjects with AIDS or with CNS opportunistic infections . Normal As seen by others , plasma The current study also shows that STI leads to increases in CSF and plasma neopterin levels and that these elevations peak later than plasma and CSF HIV RNA levels. Neopterin is an unspecific marker of immune activation that is largely derived from activated macrophages and microglia . CSF neoWhatever the underlying mechanism, the presumed axonal damage was subclinical, and no concomitant neurological deterioration was detected during the follow-up period either on the basis of symptoms or on clinical or more formal, though brief, quantitative testing. Based on this initial study, it remains unknown whether this axonal injury has clinical importance. The subject number was small and the follow-up period, for the most part, of limited duration. Hence, longer-term impact on neurological function could not be discerned, though one subject was followed for two years off treatment and did not exhibit neurological change over that time. With one exception , our suThe present study also does not establish whether axonal injury is an acute short-term event after treatment interruption or if it is more chronic. In herpes simplex type 1 (HSV-1) encephalitis, CSF NFL levels increase to a maximum approximately 8–14 days after the onset of neurological symptoms, and only slowly decrease thereafter, with abnormal levels still detected as long as 3–10 months later . SimilarThe temporal delay between viral replication (as evidenced by plasma and more particularly CSF HIV RNA levels) and the onset of axonal disturbance (measured by NFL) may also imply that Wallerian degeneration is involved in STI. If the neuronal soma is the primary site of action, the axonal changes should be a later event. A direct effect on axons in myelinated tracts or spinal roots could also be hypothesised. Alternatively, there may be a lag between HIV replication and onset of neural injury, perhaps involving immunopathological pathways that lag behind the surge in viral replication. The magnitude of the CSF viral load increase, however, did not directly predict the development of elevated CSF NFL; as noted earlier, there were subjects with high CSF viral load without change in NFL levels.In conclusion, our study raises the question of whether treatment interruption enhances the risk of brain injury. Conversely, it may also suggest that once started, continued treatment might prevent subclinical brain damage. NFL is a sensitive marker of axonal injury which in the present setting seems to disclose subclinical injury. Further studies are needed to define the frequency, duration, magnitude and pathobiological importance of this increase in CSF NFL in HIV infection.The author(s) declare that they have no competing interests.MG, LR, LH, and RWP contributed to the conception of the study, data interpretation, and writing of the paper. SGD contributed to the establishment of the clinical study and writing the paper.


A low cerebrospinal fluid (CSF) white-blood cell count (WBC) has been identified as an independent risk factor for adverse outcome in adults with bacterial meningitis. Whereas a low CSF WBC indicates the presence of sepsis with early meningitis in patients with meningococcal infections, the relation between CSF WBC and outcome in patients with pneumococcal meningitis is not understood.We examined the relation between CSF WBC, bacteraemia and sepsis in a prospective cohort study that included 352 episodes of pneumococcal meningitis, confirmed by CSF culture, occurring in patients aged >16 years.3 (interquartile range 531–6983 per mm3) and 104 patients (33%) had a CSF WBC <1000/mm3. Patients with a CSF WBC <1000/mm3 were more likely to have an unfavourable outcome  than those with a higher WBC . CSF WBC was significantly associated with blood WBC (Spearman's test 0.29), CSF protein level (0.20), thrombocyte count (0.21), erythrocyte sedimentation rate (-0.15), and C-reactive protein levels (-0.18). Patients with a CSF WBC <1000/mm3 more often had a positive blood culture  and more often developed systemic complications  than those with a higher WBC . In a multivariate analysis, advanced age , a positive blood culture , and a low thrombocyte count on admission  were associated with a CSF WBC <1000/mm3.CSF WBC was recorded in 320 of 352 episodes (91%). Median CSF WBC was 2530 per mmA low CSF WBC in adults with pneumococcal meningitis is related to the presence of signs of sepsis and systemic complications. Invasive pneumococcal infections should possibly be regarded as a continuum from meningitis to sepsis. Streptococcus pneumoniae and Neisseria meningitidis. The mortality rate was 21 percent and was higher among patients with pneumococcal meningitis than among those with meningococcal meningitis . The outcome was unfavourable in 34 percent of episodes and the strongest risk factors for an unfavourable outcome were symptoms indicative of systemic compromise, a low level of consciousness, infection with S. pneumoniae, and a low cerebrospinal fluid (CSF) white-blood cell count (WBC). The relation between unfavourable outcome and a low CSF WBC has been described previously in both patients with N. meningitidis and S. pneumoniae meningitis . In a categorization of death of patients with pneumococcal meningitis who died within 14 days after admission, the percentage of patients who died due to neurological causes was similar between groups. The percentage of patients who died due to a systemic cause was significantly smaller in the dexamethasone group than in the placebo group . The systemic complications that were potentially preventable by dexamethasone were pulmonary complications and septic shock [3, a subgroup analysis of patients included in this study with low CSF WBC on the effect of dexamethasone cannot be performed. In the current study, patients did not routinely receive corticosteroids [A recent randomised, placebo-controlled trial involving 301 adults with suspected meningitis in combination with cloudy cerebrospinal fluid, bacteria in the CSF on Gram's staining, or a CSF WBC of more than 1000 per mmatients) . In thisications . In a ca3 was a risk factor for the development of systemic complications in patients with pneumococcal meningitis. These findings suggest that a low CSF WBC may be due the presence of bacteraemia and sepsis with early meningitis and invasive pneumococcal infections should possibly be regarded as a continuum from meningitis to sepsis.We found a significant correlation between CSF WBC, blood WBC and other indexes of inflammation in the blood. In addition, a CSF WBC below 1000 per mmThe author(s) declare that they have no competing interests.MW and DvdB participated in the design and coordination of the study, performed the statistical analysis, and participated in the writing of the article. JdG and LS participated in the design and coordination of the study and in the writing of the article. JBR participated in the statistical analysis and in critical revisions of the manuscript for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The HIV/AIDS epidemic continues to spread despite more than 20 years of significant research and major advances in its treatment. The introduction of highly active antiretroviral therapy in recent years has significantly improved disease treatment with a dramatic impact in HIV/AIDS associated morbidity and mortality in countries which have access to this therapy. Despite these advances, such therapies are imperfect and other therapeutic modalities, including immune-based therapies, are being actively sought. Potential benefits of immune-based therapies include: 1) the improvement of HIV-specific immunity to enhance control of viral replication, 2) the improvement of other aspects of host immunity in order to prevent or delay the development of opportunistic infections and 3) the potential to purge virus from cellular reservoirs which are sustained despite the effects of potent antiretroviral therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been studied as one of these immune-based therapies. Several randomized, controlled trials have demonstrated benefits of using GM-CSF as an adjunct to conventional anti-retroviral therapy, although such benefits have not been universally observed. Individual studies have shown that GM-CSF increases CD4+ T cells counts and may be associated with decreased plasma HIV RNA levels. There is limited evidence that GM-CSF may help prevent the emergence of antiretroviral drug resistant viruses and that it may decrease the risk of infection in advanced HIV disease. Despite its high costs and the need to be administered subcutaneously, encouraging results continue to emerge from further studies, suggesting that GM-CSF has the potential to become an effective agent in the treatment of HIV infection. More than 20 years after its discovery, and despite extensive research in the field, HIV-1 infection remains one of the most important public health problems in the world. The HIV/AIDS epidemic continues to spread and an increasing number of people continue to live with HIV/AIDS and die from it. The advent of highly active antiretroviral therapy (HAART) marked a cornerstone in HIV/AIDS treatment that drastically changed the prognosis of HIV infection, by its ability to induce sustained suppression of viral replication . Yet HIVin vitro to enhance the activity of the antiretroviral agent zidovudine (AZT) in macrophages [In vitro, the presence of GM-CSF suppresses the expression of CXCR4 mRNA and CCR5 mRNA by monocytes differentiating in macrophages, resulting in macrophages that are relatively resistant to M-tropic HIV infection [GM-CSF is a pleiotropic growth factor that enhances the number and function of various cells from both the myeloid and lymphoid lineages, including neutrophils, monocytes and lymphocytes . It is orophages ,23 and trophages . In vitrnfection .in vitro studies that have suggested that GM-CSF enhances the action of anti-retroviral drugs (ARVD) in macrophages [in vivo. There appears to be lower frequency of resistant-mutations among subjects on zidovudine and GM-CSF, as part of their anti-retroviral regimens, versus those on AZT alone [in vitro findings and early in vivo studies, several randomized controlled trials were developed that studied the effect of GM-CSF as a treatment for HIV-1 infected individuals.In addition to rophages ,23, dataZT alone . This fiZT alone -28, the ZT alone -31. Foll10 copies/ml in the placebo group compared with 4.21 log10 in the treatment group (p = 0.29) and the mean CD4+ T cells count in the placebo group was 243 cells/mm3 compared with 178 cells/mm3 in the GM-CSF group. All subjects were on stable antiretroviral therapy, including either indinavir or ritonavir, for a mean period of 5.0 months in the placebo group and 4.8 months in the treatment group. They received either 250 μg of GM-CSF or placebo subcutaneously 3 times per week for a total of eight weeks. All subjects were followed closely every two weeks during the study and twice at week 3 and week 5 after the study ended. During the study and at both follow up time points, the viral load remained within 0.5 log10 copies/ml of the baseline values for both groups.Few randomized controlled trials of GM-CSF have shown a clear, significant reduction in HIV replication. The first randomized controlled trial on the use of GM-CSF in non-neutropenic HIV-1 infected subjects, published in 1999, did not show any significant effect of GM-CSF on plasma HIV RNA levels . This tr10 from baseline (50% vs. 10%). Since this size of a viral load decrease has been associated with clinical benefits, this study suggests that GM-CSF may have a beneficial effect in a subset of individuals.Despite no overall changes in the mean HIV RNA load between groups, more subjects in the GM-CSF group than in the control group had HIV RNA values decreased by >0.5 log3 in the control group and 50.8 cells/mm3 in the GM-CSF group. The majority of subjects entered the study with a viral load over 30000 copies/ml (62% for the placebo arm and 63% for the GM-CSF arm). There was no significant decrease in HIV-RNA in the combined strata in either the placebo or treatment group. However, GM-CSF had a positive influence on other viral parameters. GM-CSF use delayed virologic failure in those patients with plasma HIV RNA levels less than 400 copies/ml before initiation of GM-CSF therapy. At 6 months, 24 out of 29 (83%) of subjects on GM-CSF maintained viral loads below the limit of detection compared to 15 out of 28 (54%) of those on placebo (p = 0.02). This, in turn, reduced the need for antiretroviral regimen change. In this trial, ARVD regimen changes were allowed, which could have obscured a preferential decrease in viral load by GM-CSF. As such, there were fewer changes in ARVD regimens in the GM-CSF group (19%) than in the placebo group (38%) for the lower viral load stratum (p = 0.03). In the higher stratum, no significant difference in treatment change was observed . Again, this supports the idea that low-dose GM-CSF may have the potential to limit HIV replication and prevent or delay the development of drug resistant viruses, as described earlier in in vitro and in vivo studies.In the largest double blind, randomized controlled trial on GM-CSF use in HIV infected individuals published to date, 309 subjects stratified according to viral load (≤ 30000 copies/ml vs. > 30000 copies/ml) received either 250 μg of GM-CSF or placebo three times per week for 24 weeks . In tota3. Patients were excluded if they had an active AIDS defining diagnosis at the time of randomization or if they had been exposed to zidovudine for >6 months prior to study entry. The mean HIV RNA plasma levels at baseline were 93000 copies/ml in the placebo group and 155000 copies/ml in the GM-CSF group (p = 0.21). All the subjects received AZT, and 65% and 68% of subjects in the placebo and GM-CSF group respectively were also on a second agent, either ddI, ddC, 3TC or Saquinavir. Prior opportunistic infection rates were 58% in the placebo group and 70% in the treatment group (p = 0.14). This study did show a statistically significant effect of GM-CSF on viral loads. Mean HIV RNA levels declined in the GM-CSF group throughout the 6 months of the study. Over this period, the change was -0.07 log10 copies/ml in the control group as opposed to -0.60 log10copies/ml in the treatment group . As well, there was a greater number of subjects in the GM-CSF group with a decrease of 1 log10 or greater in viral load  compared with the placebo group  (p = 0.02). The reason why a decrease in viral load was observed in this study and not in other trials is unclear. It was the only trial with a smaller dose of GM-CSF  and all patients were receiving AZT, both of which might have played a role in this difference.In a Brazilian study, 105 individuals with AIDS were enrolled in a placebo-controlled, double-blind randomized control trial to receive AZT along with GM-CSF (125 μg) or placebo twice weekly for 6 months . Subject10 copies/ml) for at least 7 days prior to entry and where no HAART regimen change was allowed during the 16 weeks period of the trial. Subjects were divided in 2 groups, depending if their CD4+ T count was below or above 200 cells/mm3 at baseline and then randomized to either 250 ug of GM-CSF or placebo three times per week for 16 weeks. All patients subsequently received a 32-week course of open label GM-CSF. Baseline characteristics were similar in both groups. At baseline, in the ≥ 200 and <200 CD4+ cells/mm3 strata, median plasma RNA levels were 3.81 log10 and 4.46 log10 copies/ml, with no difference between control and treatment groups. After the 16 weeks of double-blinded treatment, the change in HIV RNA levels was +0.048 log10 copies/ml in the GM-CSF group compared with -0.103 log10 copies/ml (p = 0.036) in the placebo group, both strata combined. However, when the two strata (≥ 200 and <200 CD4+ cells/mm3) were studied individually, the changes in mean viral loads were not significant. Thus, in this trial, subjects in the GM-CSF group, irrespective of their initial CD4+ count, tended to have a modest increase in HIV RNA levels at the end of the 16-week randomized period. Although the modest increase in viral load was significant, it was not associated with a decrease in CD4 counts or an increase in clinical events, as is discussed later.More recent clinical data on the use of GM-CSF in combination with HAART continues to show some effect of GM-CSF on viral load . These d3. In both groups the viral load peaked at 6 weeks and trended down afterwards. In the GM-CSF group, the maximum viral load reached a mean of 4.97 log10 compared with 5.54 log10 in the scheduled treatment interruption-only (STI-only) group (p = 0.03). Over a period of twelve weeks, the mean area under the curve for viral loads were 47.77 log10 in the GM-CSF group and 51.88 log10 in the STI-only group (p = 0.07) This suggests not only that there is no deleterious effect of GM-CSF on plasma HIV RNA levels but that GM-CSF may help control the viral load in patients who need to stop HAART for a short period.Finally, a Swiss study evaluated the use of GM-CSF 300 μg three times a week for the first four weeks of a 12-week HAART interruption period . This smet al. is not clear. This study was somewhat unique in that it included only patients with uncontrolled viral replication. It appears likely that the impact of GM-CSF on viral load is dependant upon the setting in which GM-CSF is used. Furthermore, GM-CSF may selectively enhance the antiviral activity of specific antiretroviral agents (e.g. AZT). Also, as may be becoming apparent with other immune-based therapies, the greatest effect of GM-CSF may be observed in situations where there is the greatest degree of virologic suppression and associated immunologic recovery.Overall, the evidence regarding the effect of GM-CSF on plasma HIV-1 RNA levels is somewhat conflicting. Four of the five trials reviewed show either a significant decline or no statistical changes in the viral load. The explanation for the observed increase in viral load in the GM-CSF group in the trial by Jacobson 3 and 57 ± 58.9 cells/mm3 in the control group (p = 0.02). A significant majority (70%) of subjects treated with GM-CSF demonstrated an increase of >30% of their CD4+ T cell counts over baseline at any given time versus a minority (30%) in the placebo group (p = 0.07). When those patients with baseline CD4+ T cell counts of <50 cells/mm3 were excluded from the analysis, in order to ensure an increase of >30% was not due to daily variability, 6 of 7 patients in the GM-CSF group and 1 of 8 patients in the placebo group had a CD4+ T cell increase of >30% (p = 0.01). This may have a clinical impact as a >30% increase of the CD4+ T cell count in light of a stable viral load has been associated with a relative risk reduction of disease progression in a previous study [The initial randomized control trial of GM-CSF use in non-leukopenic HIV infected individuals, referred to previously, reported other important findings. CD4+ T cell counts reached higher levels in the treatment group, but these results did not reach statistical significance . The meaus study .An earlier randomized controlled study looking into the effect of GM-CSF on leukopenia in HIV-infected individuals also demonstrated an increase in CD4+ T cell counts. After 12 weeks of therapy (300 μg GM-CSF daily for 1 week then 150 μg twice-a-week for 11 weeks), absolute CD4+ T cell counts rose by 53% compared to baseline (p < 0.001) and was statistically different than that observed in the control group (p < 0.001) .et al, the authors reported a modest increase in the CD4+ T cell count in both groups at six months [3 compared with 12 cells/mm3 in placebo group (p = 0.42). As with the study by Skowron et al, they also observed a significant difference in the number of subjects who had a ≥ 30% increase of the CD4+ T cell count. Only 59% of subjects in the placebo group achieved this increase as opposed to 80% in the GM-CSF group (p = 0.03).Other trials observed a trend towards modest, non-significant, increases in the absolute CD4+ counts. In the study by Brites x months . In the et al., the authors reported a change in the CD4+ T cell count of +29 cells/mm3 in the GM-CSF vs. -8 cells/mm3 in the placebo group for the stratum of subjects with >200 CD4+ T cells/mm3 at baseline (p = .20) [3 stratum, with +5 cells/mm3 in the GM-CSF group at 16 weeks vs. -5 cells/mm3 in the placebo group but this did not reach statistical significance (p = 0.22).In the other, more recent trial, from Jacobson p = .20) . They ob6 cells/L for the placebo group and 50.8 × 106 cells/L for the treatment group. At 12 months the mean CD4+ T cell count was 102 ± 15 × 106 cells/L in the placebo group vs. 152 ± 18 × 106 cells/L in the treatment group. This was also reflected by statistically significantly greater increases in the CD4+ T cell count at 1, 3 and 6 months in the GM-CSF group.Fairly convincing evidence of a significant increase in absolute CD4+ leukocyte count following treatment with GM-CSF comes from the phase III randomized control trial by Angel and colleagues, described earlier . The basIt might be speculated that, since this was the largest (n = 307) and one of the longest (24 weeks) randomized control trial of GM-CSF use in HIV infected individuals, it maybe the only study with enough power to demonstrate statistical significance. As other randomized control trials show trends towards higher CD4+ T cell counts in the GM-CSF group, and statistically significant difference in various sub-analysis, it is possible that an appropriately conducted meta-analysis would clarify the impact of GM-CSF on CD4+ T cell counts.6 cells/L at baseline to 537 × 106 cells/L at four weeks after stopping HAART in the STI-only group (p < 0.001). In the GM-CSF treated group, there was no significant change in the CD4+ T cell counts four weeks after stopping HAART. CD4+ T cell counts were 890 × 106 cells/L at baseline and 792 × 106 cells/L at week four (p = 0.6). This adds evidence that GM-CSF could have a beneficial effect on CD4+ T cell counts.The recent study by the Swiss group also supports a positive effect of GM-CSF on CD4+ lymphocytes count . In thatet al, a total of 4 patients had to discontinue GM-CSF use because of toxicity or acute allergic reactions [GM-CSF has an excellent safety and tolerability profile when used in HIV-1 infected individuals ,15-17. Ieactions ,15,16. Tet al. has been the only study to use clinical events as endpoints, using the Centers for Diseases Control and Prevention definition of opportunistic infections (OI), bacterial pneumonia or death as their primary endpoint [The large phase III trial by Angel endpoint . An effeet al. did noticed that all 17 subjects in the GM-CSF arm who developed an OI had a prior history of one or more of these infections. In the placebo group, only 50% of the 14 subjects who developed an OI during the study had a prior history of OI (p < 0.01). This might prove to be an important role for adjunct treatment with GM-CSF as OI are still an important cause of morbidity and mortality in HIV infected individuals.For individuals who do not have a history of OI, GM-CSF may decrease the risk of a first opportunistic infection. Despiteet al. did show a non-significant reduction in clinical events in the GM-CSF group [The most recent randomized control trail by Jacobson SF group . No HIV et al. failed to show any impact of GM-CSF on clinical events during HAART interruption. However, they studied only 33 patients with high CD4 counts and off HAART for a limited period of time [Finally, the study by Fagard  of time .Despite all these results, questions still remain as to whether use of GM-CSF is associated with a reduction in the incidence of AIDS related morbidity and mortality, as even the authors of the largest phase III study published to date admit to a lack of power in their trial . The intIn various studies GM-CSF has a positive effect on important parameters of HIV infection, namely plasma HIV RNA levels and CD4+ lymphocytes counts. Although the positive effects are modest and not universally observed, they are significant in many trials. Moreover, the positive effect on these measures may translate into significant clinical benefits. Clinical outcome results of current randomized controlled trials are, thus far, somewhat encouraging. Despite the frequent lack of statistical significance, there are positive trends towards clinical benefit of GM-CSF use in these studies. Moreover, the largest randomized control trial did show that GM-CSF produces a significant reduction in the time to first infection or death. The possibility of allowing longer disease free periods is a desirable outcome for HIV infected individuals, contributing to improved quality of life. However, the high cost of GM-CSF and its mode of administration may be difficult hurdles for patients to overcome.Future trials designed to look at specific clinical outcomes, for example diseases free period, progression of HIV infection and quality of life, might bring to light additional beneficial effects of GM-CSF. This would require focusing on patients with advanced HIV disease and lower CD4 counts. Alternatively, future trials could focus on the use of GM-CSF as an adjuvant therapy, either to HAART or as an adjuvant with HIV or other vaccines. There is growing evidence that GM-CSF enhances the immune response to vaccines directed against both infectious agents and various cancers . ClinicaIt remains to be seen if GM-CSF will ever loose its experimental status and become an accepted therapy for selected individuals HIV infection. The evidence for the role of immunotherapy in HIV/AIDS is ever increasing and GM-CSF might very well become a widely accepted treatment in the years to come.The author(s) declare that they have no competing interests.Table 1 is a summary of clinical trials of GM-CSF in the treatment of HIV infection.Click here for file


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