Role of the inhibitory KIR ligand HLA-Bw4 and HLA-C expression levels in the recognition of leukemic cells by Natural Killer cells

Transplantation of acute myeloid leukemia (AML) patients with grafts from related haploidentical donors has been shown to result in a potent graft-versus-leukemia effect. This effect is mediated by NK cells because of the lack of activation of inhibitory killer cell immunoglobulin-like receptors (KIRs) which recognize HLA-Bw4 and HLA-C alleles. However, conflicting results have been reported about the impact of KIR ligand mismatching on the outcome of unrelated HLA-mismatched hematopoietic stem cells transplants (HSCT) to leukemic patients. The interpretation of these conflicting results is hampered by the scant information about the level of expression of HLA class I alleles on leukemic cells, although this variable may affect the activation of inhibitory KIRs. Therefore in the present study, utilizing a large panel of human monoclonal antibodies we have measured the level of expression of HLA-A, -B and -C alleles on 20 B-chronic lymphoid leukemic (B-CLL) cell preparations, on 16 B-acute lymphoid leukemic (B-ALL) cell preparations and on 19 AML cell preparations. Comparison of the level of HLA class I antigen expression on leukemic cells and autologous normal T cells identified selective downregulation of HLA-A and HLA-B alleles on 15 and 14 of the 20 B-CLL, on 2 and 5 of the 16 B-ALL and on 7 and 11 of the 19 AML patients tested, respectively. Most interestingly HLA-C alleles were markedly downregulated on all three types of leukemic cells; the downregulation was most pronounced on AML cells. The potential functional relevance of these abnormalities is suggested by the dose-dependent enhancement of NK cell activation caused by coating the HLA-HLA-Bw4 epitope with monoclonal antibodies on leukemic cells which express NK cell activating ligands. Our results suggest that besides the HLA and KIR genotype, expression levels of KIR ligands on leukemic cells should be included among the criteria used to select the donor-recipient combinations for HSCT.     

Polymorphous exudates and atypical mononuclear cells in the cerebrospinal fluid of patients with Sj?gren's syndrome

Central nervous system (CNS) complications of Sj?gren's syndrome are now well recognized. To determine if any of the pathologic changes in the CNS in patients with Sj?gren's syndrome were reflected in the cellular composition of cerebrospinal fluid (CSF), we examined the CSF of 14 patients with Sj?gren's syndrome and neurologic symptoms and compared the differential cell counts in those cases with those of 14 control patients with similar neurologic symptoms. Patients with Sj?gren's syndrome had polymorphous (mixed) inflammatory exudates in CSF, composed predominantly of lymphocytes, but including variable numbers of plasma cells, neutrophils and erythrocytes. In addition, the CSF of all patients with Sj?gren's syndrome contained large, atypical, morphologically distinct mononuclear cells. The mean percentage of these cells in the CSF of patients with Sj?gren's syndrome (8.3 +/- 1.9) was significantly higher (p less than 0.001) than that observed in the control patients (0.7 +/- 0.2). These results suggest that involvement by Sj?gren's syndrome may be suspected by noting a polymorphous exudate containing characteristic atypical mononuclear cells in CSF obtained by lumbar puncture.     

Herpesvirus-Associated Central Nervous System Diseases after Allogeneic Hematopoietic Stem Cell Transplantation

Herpesvirus infections of the central nervous system (CNS) are associated with encephalitis/myelitis and lymphoproliferative diseases in immunocompromised individuals. As of now, data of herpesvirus-associated CNS diseases in transplant recipients is limited. Hence, in this prospective study, we investigated the incidence of herpesvirus-associated CNS diseases and explored the diagnosis of these diseases in 281 allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Herpesvirus-DNA and cerebrospinal fluid (CSF) cells were sampled from 58 recipients with herpesvirus-associated diseases or with unexplainable CNS manifestations. Results showed that 23 patients were diagnosed as herpesvirus-associated CNS diseases, including 15 Epstein-Barr virus (EBV)-associated diseases (4 encephalitis and 11 lymphoproliferative diseases), 5 herpes simplex virus type 1 encephalitis, 2 cytomegalovirus encephalitis/myelitis and 1 varicella zoster virus encephalitis. The median time of diseases onset was 65 (range 22-542) days post-transplantation. The 3-year cumulative incidence of herpesvirus-associated encephalitis/myelitis and post-transplant lymphoproliferative disorder (PTLD) was 6.3% ±1.9% and 4.1% ±1.2%, respectively. Of the evaluable cases, CSF cells mainly consisted of CD19⁺CD20⁺ B cells (7/11) and had clonal rearrangement of immunoglobulin genes (3/11) in patients with CNS-PTLD. On the contrary, in patients with encephalitis/myelitis, CSF cells were comprised of different cell populations and none of the gene rearrangement was detected. Herpesvirus-associated CNS diseases are common in the early stages of allo-HSCT, wherein EBV is the most frequent causative virus. The immunophenotypic and clonal analysis of CSF cells might be helpful in the differential diagnosis between encephalitis and lymphoproliferative diseases.     

Increased levels of tumor necrosis alpha and soluble vascular endothelial adhesion molecule-1 in the cerebrospinal fluid of patients with connective tissue diseases and multiple sclerosis

The aim of the present study was to investigate the serum and cerebrospinal fluid (CSF) concentrations of tumor necrosis factor alpha (TNF-alpha) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in patients with primary progressive form of multiple sclerosis (MS) and in patients with connective tissue diseases (CTDs) complicated with central nervous system (CNS) involvement. Stimulation of sVCAM-1 release by TNF-alpha was demonstrated on endothelial cells of brain vessels. We intended to present the TNF-alpha stimulated elevation of sVCAM-1 in the serum and CSF in any cases of CNS lesion. Fifty patients with several CTDs complicated with neuropsychiatric symptoms and 25 MS patients with primary chronic progressive form of the disease were selected. Determinations of TNF-alpha and sVCAM-1 were performed using ELISA methods. TNF-alpha and sVCAM-1 concentrations were elevated in the CSF of all patients, intrathecal synthesis of sVCAM-1 was demonstrated in MS patients. The changes in the TNF-alpha and sVCAM-1 concentrations were independent from the clinical manifestations, immunoserological changes and quality of neuropsychiatric symptoms of the CTDs. The stimulatory effect of TNF-alpha was more pronounced in the CSF of MS patients.     

Intrathecal levels of matrix metalloproteinases in systemic lupus erythematosus with central nervous system engagement

Symptoms originating from the central nervous system (CNS) occur frequently in patients with systemic lupus erythematosus (SLE), and CNS involvement in lupus is associated with increased morbidity and mortality. We recently showed that neurones and astrocytes are continuously damaged during the course of CNS lupus. The matrix metalloproteinases (MMPs) are a group of tissue degrading enzymes that may be involved in this ongoing brain destruction. The aim of this study was to examine endogenous levels of free, enzymatically active MMP-2 and MMP-9 in cerebrospinal fluid from patients with SLE. A total of 123 patients with SLE were evaluated clinically, with magnetic resonance imaging of brain and cerebrospinal fluid (CSF) analyses. Levels of free MMP-2 and MMP-9 were determined in CSF using an enzymatic activity assay. CSF samples from another 22 cerebrally healthy individuals were used as a control. Intrathecal MMP-9 levels were significantly increased in patients with neuropsychiatric SLE as compared with SLE patients without CNS involvement (P < 0.05) and healthy control individuals (P = 0.0012). Interestingly, significant correlations between MMP-9 and intrathecal levels of neuronal and glial degradation products were noted, indicating ongoing intrathecal degeneration in the brains of lupus patients expressing MMP-9. In addition, intrathecal levels of IL-6 and IL-8 – two cytokines that are known to upregulate MMP-9 – both exhibited significant correlation with MMP-9 levels in CSF (P < 0.0001), suggesting a potential MMP-9 activation pathway. Our findings suggest that proinflammatory cytokine induced MMP-9 production leads to brain damage in patients with CNS lupus.     

Cytodiagnosis of neoplasms of the central nervous system in cerebrospinal fluid samples with an application of selective immunostains in differentiation.

In this study cytological findings in specimens of cerebrospinal fluid (CSF) of central nervous system (CNS) tumours (16 primaries, 57 metastatic and 12 suspicious) are presented, which were diagnosed over a period of 7 years in 85 patients (50 females and 35 males) with an age range of 2-76 years. The follow-up included information from clinicians and a review of medical charts, histological correlation and/or further investigations following cytodiagnosis. The patients clinically presented with signs and symptoms of meningeal involvement. The primary tumours included six medulloblastomas, eight gliomas (four glioblastomata multiforme, two anaplastic astrocytomas, and two ependymomas) and two germinomas. The metastatic tumours were 14 melanomas, 19 breast carcinomas, four leukaemias, six B-cell lymphomas, five adenocarcinomas of gastrointestinal origin, seven carcinomas of lung, one retinoblastoma and one neuroblastoma. Twelve cases were reported as suspicious. On further investigations, four of these were from a primary tumour (two glioblastomata multiforme and two anaplastic astrocytomas) while the other eight cases were of a metastasis (one B-cell lymphoma, three breast carcinomas, three melanomas and one adenocarcinoma of gastrointestinal origin). Using a panel of selective immunostains in some of the cases supported the cytological diagnosis and this was considered useful in furthering cytodiagnosis. In 75 of the patients the CSF samples were obtained on a spinal tap while in 10 patients the samples were received as ventricular CSF. There were no false-positive cases. The results of our study suggest that CSF cytology in the diagnosis of CNS tumours is quite reliable and reflects involvement of leptomeninges or the ventricles. Furthermore, the use of selective immunostains can be helpful in confirming the cytological impression and source of the tumour.     

Cerebrospinal Fluid Cytokine Profiles Predict Risk of Early Mortality and Immune Reconstitution Inflammatory Syndrome in HIV-Associated Cryptococcal Meningitis

Understanding the host immune response during cryptococcal meningitis (CM) is of critical importance for the development of immunomodulatory therapies. We profiled the cerebrospinal fluid (CSF) immune-response in ninety patients with HIV-associated CM, and examined associations between immune phenotype and clinical outcome. CSF cytokine, chemokine, and macrophage activation marker concentrations were assayed at disease presentation, and associations between these parameters and microbiological and clinical outcomes were examined using principal component analysis (PCA). PCA demonstrated a co-correlated CSF cytokine and chemokine response consisting primarily of Th1, Th2, and Th17-type cytokines. The presence of this CSF cytokine response was associated with evidence of increased macrophage activation, more rapid clearance of Cryptococci from CSF, and survival at 2 weeks. The key components of this protective immune-response were interleukin (IL)-6 and interferon-γ, IL-4, IL-10 and IL-17 levels also made a modest positive contribution to the PC1 score. A second component of co-correlated chemokines was identified by PCA, consisting primarily of monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α). High CSF chemokine concentrations were associated with low peripheral CD4 cell counts and CSF lymphocyte counts and were predictive of immune reconstitution inflammatory syndrome (IRIS). In conclusion CSF cytokine and chemokine profiles predict risk of early mortality and IRIS in HIV-associated CM. We speculate that the presence of even minimal Cryptococcus-specific Th1-type CD4+ T-cell responses lead to increased recruitment of circulating lymphocytes and monocytes into the central nervous system (CNS), more effective activation of CNS macrophages and microglial cells, and faster organism clearance; while high CNS chemokine levels may predispose to over recruitment or inappropriate recruitment of immune cells to the CNS and IRIS following peripheral immune reconstitution with ART. These results provide a rational basis for future studies of immune modulation in CM, and demonstrate the potential of baseline immune profiling to identify CM patients most at risk of mortality and subsequent IRIS.     

Interleukin-34 Restores Blood–Brain Barrier Integrity by Upregulating Tight Junction Proteins in Endothelial Cells

Interleukin-34 (IL-34) is a newly discovered cytokine as an additional ligand for colony stimulating factor-1 receptor (CSF1R), and its functions are expected to overlap with colony stimulating factor-1/macrophage-colony stimulating factor. We have previously shown that the IL-34 is primarily produced by neurons in the central nervous system (CNS) and induces proliferation and neuroprotective properties of microglia which express CSF1R. However, the functions of IL-34 in the CNS are still elucidative. Here we show that CNS capillary endothelial cells also express CSF1R. IL-34 protected blood–brain barrier integrity by restored expression levels of tight junction proteins, which were downregulated by pro-inflammatory cytokines. The novel function of IL-34 on the blood–brain barrier may give us a clue for new therapeutic strategies in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis and Alzheimer''s disease.     

Elevation of Matrix Metalloproteinase-9 Level in Cerebrospinal Fluid of Tick-Borne Encephalitis Patients Is Associated with IgG Extravassation and Disease Severity

Background

Tick-borne encephalitis (TBE), caused by tick-borne encephalitis virus (TBEV), is an infectious disease involving the central nervous system (CNS). The pathogenesis of CNS injury has not been clearly demonstrated. Matrix metalloproteinase-9 (MMP-9) and some cytokines, such as interleukin 6 (IL-6), may play important roles in the disruption of the blood-brain barrier (BBB) and the pathogenesis of TBE.

Methods

72 cerebrospinal fluid (CSF) samples were collected from TBE patients in north eastern China. IgG levels in CSF and serum were compared and MMP-9 and IL-6 levels were evaluated by ELISA. The correlation between the elevated MMP-9 levels and IgG extravasation, disease severity, and neuroinflammation was analyzed.

Results

Increased concentration of MMP-9 was detected in some of the CSF samples, and the elevation was found to be closely related to CSF TBEV IgG extravasation and enhancement of IL-6 expression. Moreover, elevated levels of MMP-9 were found to be correlated with IL-6 enhancement. Four of the 72 patients, the ones who died, presented with high CSF MMP-9 levels.

Conclusions

In TBE patients, elevated CSF MMP-9 levels were associated with brain inflammatory reaction, disruption of the blood-brain barrier, and disease severity.     

B Cell,Th17, and Neutrophil Related Cerebrospinal Fluid Cytokine/Chemokines Are Elevated in MOG Antibody Associated Demyelination

Background

Myelin oligodendrocyte glycoprotein antibody (MOG Ab) associated demyelination represents a subgroup of autoimmune demyelination that is separate from multiple sclerosis and aquaporin 4 IgG-positive NMO, and can have a relapsing course. Unlike NMO and MS, there is a paucity of literature on immunopathology and CSF cytokine/chemokines in MOG Ab associated demyelination.

Aim

To study the differences in immunopathogenesis based on cytokine/chemokine profile in MOG Ab-positive (POS) and -negative (NEG) groups.

Methods

We measured 34 cytokines/chemokines using multiplex immunoassay in CSF collected from paediatric patients with serum MOG Ab POS [acute disseminated encephalomyelitis (ADEM = 8), transverse myelitis (TM = 2) n = 10] and serum MOG Ab NEG (ADEM = 5, TM = 4, n = 9) demyelination. We generated normative data using CSF from 20 non-inflammatory neurological controls.

Results

The CSF cytokine and chemokine levels were higher in both MOG Ab POS and MOG Ab NEG demyelination groups compared to controls. The CSF in MOG Ab POS patients showed predominant elevation of B cell related cytokines/chemokines (CXCL13, APRIL, BAFF and CCL19) as well as some of Th17 related cytokines (IL-6 AND G-CSF) compared to MOG Ab NEG group (all p<0.01). In addition, patients with elevated CSF MOG antibodies had higher CSF CXCL13, CXCL12, CCL19, IL-17A and G-CSF than patients without CSF MOG antibodies.

Conclusion

Our findings suggest that MOG Ab POS patients have a more pronounced CNS inflammatory response with elevation of predominant humoral associated cytokines/chemokines, as well as some Th 17 and neutrophil related cytokines/chemokines suggesting a differential inflammatory pathogenesis associated with MOG antibody seropositivity. This cytokine/chemokine profiling provides new insight into disease pathogenesis, and improves our ability to monitor inflammation and response to treatment. In addition, some of these molecules may represent potential immunomodulatory targets.     

Stage Progression and Neurological Symptoms in Trypanosoma brucei rhodesiense Sleeping Sickness: Role of the CNS Inflammatory Response

Background

Human African trypanosomiasis progresses from an early (hemolymphatic) stage, through CNS invasion to the late (meningoencephalitic) stage. In experimental infections disease progression is associated with neuroinflammatory responses and neurological symptoms, but this concept requires evaluation in African trypanosomiasis patients, where correct diagnosis of the disease stage is of critical therapeutic importance.

Methodology/Principal Findings

This was a retrospective study on a cohort of 115 T.b.rhodesiense HAT patients recruited in Eastern Uganda. Paired plasma and CSF samples allowed the measurement of peripheral and CNS immunoglobulin and of CSF cytokine synthesis. Cytokine and immunoglobulin expression were evaluated in relation to disease duration, stage progression and neurological symptoms. Neurological symptoms were not related to stage progression (with the exception of moderate coma). Increases in CNS immunoglobulin, IL-10 and TNF-α synthesis were associated with stage progression and were mirrored by a reduction in TGF-β levels in the CSF. There were no significant associations between CNS immunoglobulin and cytokine production and neurological signs of disease with the exception of moderate coma cases. Within the study group we identified diagnostically early stage cases with no CSF pleocytosis but intrathecal immunoglobulin synthesis and diagnostically late stage cases with marginal CSF pleocytosis and no detectable trypanosomes in the CSF.

Conclusions

Our results demonstrate that there is not a direct linkage between stage progression, neurological signs of infection and neuroinflammatory responses in rhodesiense HAT. Neurological signs are observed in both early and late stages, and while intrathecal immunoglobulin synthesis is associated with neurological signs, these are also observed in cases lacking a CNS inflammatory response. While there is an increase in inflammatory cytokine production with stage progression, this is paralleled by increases in CSF IL-10. As stage diagnostics, the CSF immunoglobulins and cytokines studied do not have sufficient sensitivity to be of clinical value.     

Therapeutic implications of serum factors inhibiting proliferation and inducing differentiation of myeloid leukemic cells

Murine post-endotoxin sera contain high levels of myeloid colony-stimulating factor(s) (GM-CSF) and factors capable of inducing terminal granulocyte and macrophage differentiation of the murine myelomonocytic leukemic cell line WEHI-3. The combination of C. parvum and endotoxin induced a serum activity capable of inducing tumor necrosis and inhibiting leukemic colony formation in vitro. This factor (TNF) could be separated from the differentiation-inducing factor (GM-DF) and from CSF. In conjunction with a Phase I trial of highly purified endotoxin in patients with advanced malignancy, we monitored human post-endotoxin sera for CSF and GM-DF. Induction of GM-DF occurred maximally at 2-6 h and was associated with increased serum levels of CSF active against the patient's own bone marrow. Following repeated injections of escalating doses of endotoxin, persistent levels of GM-DF were detected both pre-endotoxin and 24 h post-endotoxin treatment. The ability to induce repeatedly a serum protein with potent capacity to promote terminal differentiation of myelomonocytic leukemic cells suggests a possible therapeutic role in human myeloid leukemias.     

Alzheimer's disease cerebrospinal fluid antibodies display selectivity for microglia

Previous investigations demonstrated that the cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients contains antibodies that recognize specific neuronal populations in the adult rat central nervous system (CNS). These findings suggest a pathogenic role for immunological aberrations in this disorder. To determine if antibodies may provide a means to differentially diagnose the dementias, CSF from a diversified dementia population was screened against the developing rat CNS and a cell culture system. Markings produced by AD CSF were distinctly different from those of vascular dementias (VAD) against the developing rat CNS. More importantly, some AD CSF recognized amoeboid microglia. The recognition of amoeboid microglia by antibodies in AD CSF is particularly interesting since these cells proliferate in response to nervous system disease and also engulf debris. A cell culture technique was developed to allow the rapid screening of CSF antibodies. Patient CSF produced five different types of markings in the cell culture: microglia, glioblasts, fibers, nonspecific, or negative. Correlations with these structures and the diagnosis of four different dementia populations revealed that, in comparison to the other groups, AD CSF displayed remarkable selectivity toward microglial cells. Cortical biopsies from patients suspected to have AD were incubated with the patient's own CSF and that of confirmed AD patients. Both CSF samples recognized microglial cells in the patient's cortical biopsy. The same CSF samples incubated against normal human cortical autopsy or a biopsy from a 3-mo-old child displayed negative immunoreactivity. These three approaches suggest that the presence of CSF microglial antibodies may be a means to distinguish AD patients from other dementias. The results add further support to the widely growing concept that inflammation and similar immune mechanisms may contribute to AD pathogenesis.     

Detection of cytomegalovirus in infant cerebrospinal fluid by conventional PCR, nested PCR and PCR-Digene

The possibility of amplification of human cytomegalovirus (HCMV) DNA in cerebrospinal fluid (CSF) for the diagnosis of HCMV central nervous system (CNS) infection in infants was studied. Single-step PCR, nested PCR and PCR-Digene were used to assay CSF specimens from 37 patients. Criteria for patient inclusion in the study were: 1. clinical manifestations suggesting CMV neuroinfection such as seizures, hypertonia, hypotonia, intracranial calcification, microcephaly, chorioretinitis; 2. any of the following symptoms: anaemia, hepetomegaly, prolonged cholestatic jaundice, or hepatitis, splenomegaly, thrombocytopenia, intrauterine hypotrophy; 3. serologic presentation, and/or positive results for CMV infection obtained by single-step PCR and PCR-Digene in urine and/or blood. PCR-Digene results were positive in 6 CSF samples. Four CSF samples were positive by nested PCR and 1 CSF sample by single step PCR. We found that the double PCR was about ten or more times more sensitive than single PCR and the PCR-Digene was only three times more sensitive than nested-PCR. The results were correlated with serology. Thirty-three out of 37 examined patients were seropositive (ELISA IgG); ELISA IgM gave positive results in 9 patients. In control studies, cells infected with other members of the herpes virus family were negative with these methods, which suggest that amplification combined with primers from the IE and the L-region of CMV is specific. In conclusion, nested-PCR seems to be the best method for early diagnosis of CMV infection in CSF due to an absence of false positive results and its high specificity and sensitivity.     

A link between chemokine levels and disease severity in human African trypanosomiasis

Trypanosoma brucei gambiense infection is an important public health challenge in sub-Saharan Africa. This parasitic disease is difficult to diagnose due to insidious clinical signs and transient parasitaemias. The clinical course is marked by two stages of increasing disease severity. An early systemic parasitic invasion is followed by the development of a progressive meningo-encephalitis. During this latter stage, a broad spectrum of neurological signs appears, which finally lead to a demyelinating and fatal stage if untreated. Treatment is toxic and difficult to administer when the CNS is invaded. Therefore, accurate diagnostic methods for stage determination are needed. The classically used criteria are not sufficiently specific and mechanisms of parasite invasion through the blood-brain barrier remain poorly understood. As cytokines/chemokines are involved in the early recruitment of leukocytes into the CNS, this study has focused on their potential value to define the onset of CNS involvement. Levels of monocyte chemoattractant protein-1/CCL-2, macrophage inflammatory protein-1α/CCL-3, IL-8/CXCL-8, regulated upon activation T cell expressed and secreted (RANTES)/CCL-5 and IL-1β were measured in paired sera and CSF from 57 patients and four controls. Patients were classified into three groups (stage 1, intermediate and stage 2) according to current field criteria for stage determination (CSF cell count, presence of trypanosomes in CSF and neurological signs). In sera, cytokine/chemokine levels were poorly related to disease stage. Only CXCL-8 was higher in stage 1 patients when compared with stage 2 and CCL-5 was higher in controls when compared with patients. In contrast, in CSF the expression of the selected cytokines, except CCL-5, was associated with the presence of neurological signs, demonstrating their diagnostic value. We observed a relationship between the presence of trypanosomes or trypanosome-related compounds in CSF and levels of IL-1β, CXCL-8, CCL-2 and CCL-3. These cytokines and chemokines may be triggered by the parasite and hence are potential markers of CNS invasion.     

Central Nervous System Compartmentalization of HIV-1 Subtype C Variants Early and Late in Infection in Young Children

HIV-1 subtype B replication in the CNS can occur in CD4+ T cells or macrophages/microglia in adults. However, little is known about CNS infection in children or the ability of subtype C HIV-1 to evolve macrophage-tropic variants. In this study, we examined HIV-1 variants in ART-naïve children aged three years or younger to determine viral genotypes and phenotypes associated with HIV-1 subtype C pediatric CNS infection. We examined HIV-1 subtype C populations in blood and CSF of 43 Malawian children with neurodevelopmental delay or acute neurological symptoms. Using single genome amplification (SGA) and phylogenetic analysis of the full-length env gene, we defined four states: equilibrated virus in blood and CSF (n = 20, 47%), intermediate compartmentalization (n = 11, 25%), and two distinct types of compartmentalized CSF virus (n = 12, 28%). Older age and a higher CSF/blood viral load ratio were associated with compartmentalization, consistent with independent replication in the CNS. Cell tropism was assessed using pseudotyped reporter viruses to enter a cell line on which CD4 and CCR5 receptor expression can be differentially induced. In a subset of compartmentalized cases (n = 2, 17%), the CNS virus was able to infect cells with low CD4 surface expression, a hallmark of macrophage-tropic viruses, and intermediate compartmentalization early was associated with an intermediate CD4 entry phenotype. Transmission of multiple variants was observed for 5 children; in several cases, one variant was sequestered within the CNS, consistent with early stochastic colonization of the CNS by virus. Thus we hypothesize two pathways to compartmentalization: early stochastic sequestration in the CNS of one of multiple variants transmitted from mother to child, and emergence of compartmentalized variants later in infection, on average at age 13.5 months, and becoming fully apparent in the CSF by age 18 months. Overall, compartmentalized viral replication in the CNS occurred in half of children by year three.     

Th1/Th2 cytokine balance and nitric oxide in cerebrospinal fluid and serum from patients with multiple sclerosis

Tumor necrosis factor (TNF-alpha) and IL-10 are key regulators of the T helper (Th)1/Th2 balance, which is critically skewed in many pathological conditions including immune-mediated inflammatory diseases of central nervous system (CNS) such as multiple sclerosis (MS). Nitric oxide (NO) has been reported to have dual effects on CNS pathology, and to play an important role in MS. We performed a cross-sectional study in 17 randomly selected patients during MS flare-up, and compared levels of TNF-alpha, IL-10 and NO in serum and cerebrospinal fluid (CSF) with the serum values of these mediators in two different control groups, healthy subjects and HIV-infected untreated patients. Serum and CSF values of TNF-alpha, IL-10 and NO were higher in MS patients than in the serum of healthy controls. Two MS patients showed increased levels of NO in CSF, with inversion of the NO(SERUM)/NO(CSF) quotient, which is clearly indicative of an intrathecal production of NO. No correlation among the values of both cytokines and NO, and the laboratory parameters analysed in MS patients (IgG index, presence of IgG oligoclonal bands and albumin quotient) was found. The high levels of TNF-alpha and IL-10 (both in serum and CSF) accompanying an MS attack suggest a simultaneous expression of Th1 and Th2 cytokines as opposed to sequential expression of Th1 followed by Th2 as described in the models of experimental autoimmune encephalomyelitis (EAE). Globally, our results support the inherent heterogeneity of the disease.     

The basics of preclinical drug development for neurodegenerative disease indications

Background

Because of the emerging intersections of HIV infection and Alzheimer's disease, we examined cerebrospinal fluid (CSF) biomarkers related of amyloid and tau metabolism in HIV-infected patients.

Methods

In this cross-sectional study we measured soluble amyloid precursor proteins alpha and beta (sAPPα and sAPPβ), amyloid beta fragment 1-42 (Aβ_1-42), and total and hyperphosphorylated tau (t-tau and p-tau) in CSF of 86 HIV-infected (HIV+) subjects, including 21 with AIDS dementia complex (ADC), 25 with central nervous system (CNS) opportunistic infections and 40 without neurological symptoms and signs. We also measured these CSF biomarkers in 64 uninfected (HIV-) subjects, including 21 with Alzheimer's disease, and both younger and older controls without neurological disease.

Results

CSF sAPPα and sAPPβ concentrations were highly correlated and reduced in patients with ADC and opportunistic infections compared to the other groups. The opportunistic infection group but not the ADC patients had lower CSF Aβ_1-42 in comparison to the other HIV+ subjects. CSF t-tau levels were high in some ADC patients, but did not differ significantly from the HIV+ neuroasymptomatic group, while CSF p-tau was not increased in any of the HIV+ groups. Together, CSF amyloid and tau markers segregated the ADC patients from both HIV+ and HIV- neuroasymptomatics and from Alzheimer's disease patients, but not from those with opportunistic infections.

Conclusions

Parallel reductions of CSF sAPPα and sAPPβ in ADC and CNS opportunistic infections suggest an effect of CNS immune activation or inflammation on neuronal amyloid synthesis or processing. Elevation of CSF t-tau in some ADC and CNS infection patients without concomitant increase in p-tau indicates neural injury without preferential accumulation of hyperphosphorylated tau as found in Alzheimer's disease. These biomarker changes define pathogenetic pathways to brain injury in ADC that differ from those of Alzheimer's disease.     

Experiences with the Ommaya reservoir for prophylaxis and treatment of the central nervous system in adult acute myelocytic leukemia

Intraventricular chemotherapy was administered to adult AML patients via an Ommaya reservoir. Twenty-eight patients received central nervous system (CNS) prophylaxis and seven patients were treated for meningeal leukemia (ML). A treatment course lasted at least 6 months. Asymptomatic ML developed in two patients (7%) of the prophylaxis group concomitantly with bone marrow relapse. One of these patients had not completed the standard course. CNS remission could be obtained in all evaluable patients with ML. The easy entrance to the cerebrospinal fluid (CSF) offers the advantage of frequent investigations of the CSF, early diagnosis and treatment of CNS relapses without radiotherapy, and caused little patient discomfort. CNS prophylaxis in this small study seemed to prolong first remission duration slightly. In M4 and M5 subtypes CNS prophylaxis can be valuable.