Correlations between cerebrospinal fluid biomarkers, neurocognitive tests, and resting-state electroencephalography (rsEEG) in patients with HIV-associated neurocognitive disorders.

HIV-associated neurocognitive disorders (HAND) are highly prevalent in people living with HIV (PLWH) despite successful treatment with combination antiretroviral therapy (cART). HAND pathogenesis is complex and definitive surrogate biomarkers are not clearly defined. Brain function has been assessed through the evaluation of cortical source rhythms with delta waves associated with neurological impairment. The aim of this study was to assess the correlation between EEG cortical sources, cerebrospinal fluid (CSF) biomarkers, and neurocognitive tests in PLWH with HAND. PLWH with HAND without significant comorbidities were enrolled. Baseline rsEEG-LORETA waves, CSF biomarkers (t-tau, p-tau, ß-amiloid42, neopterin, S100ß), and neurocognitive tests were correlated and compared through non-parametric tests (Spearman's rho and Mann-Whitney); data are presented as medians (interquartile ranges). Fifty-four patients were enrolled. Median time of suppressed HIV-RNA and CD4+ T-lymphocyte were 10 years (5.5-15) and 691/uL (477-929). Thirty-nine participants (72%) underwent CSF collection: abnormal biomarkers were found in a small percentage. Only neopterin showed a statistically significant correlation with delta activity [parietal (rho 0.579; p < 0.001), occipital (rho 0.493; p = 0.007), and global sources (rho 0.464 p = 0.011)]. Seven patients (12.9%) showed an abnormal neopterin level (> 1.5 ng/mL) with significantly higher delta source activity compared to the ones with in-range concentrations. We observed a statistically significant correlation between working memory test Trail Making B with both CSF neopterin levels and delta waves (p values < 0.05). In a small sample of PLWH with HAND, we observed that higher CSF neopterin levels were associated with higher EEG delta waves and worse working memory tests.

Clinical and genetic factors associated with kidney tubular dysfunction in a real-life single centre cohort of HIV-positive patients.

BACKGROUND: Tenofovir (TDF) is one of the most widely used antiretroviral drug. Despite the high degree of tolerability a small percentage of patients experienced alteration in tubular function during TDF use. Intracellular TDF disposition is regulated by ATP-binding cassette (ABC) drug efflux transporters and, a reduced transport activity may be implicated in accumulation of TDF into the cells. The aim of our study was to assess the major determinants of TDF associated tubular dysfunction (KTD) in a real-life setting including the usefulness of single-nucleotide polymorphisms (SNPs) mapping into ABCC2, ABCC4 and ABCC10 genes. METHODS: We retrospectively analyzed all HIV positive patients who were followed at the Infectious Diseases Unit, DIBIC Luigi Sacco, University of Milan from April 2013 to June 2016. All patients treated with TDF who underwent a genotypization for the functional variants mapping in ABCC2 rs717620 (-24 C > T), ABCC4 rs1751034 (3463 A > G) and ABCC10 rs2125739 (T > C) were evaluated. KTD was defined as the presence of urine phosphate wasting and/or proteinuria at 24 h urine analysis. RESULTS: One hundred fifty-eight patients were genotyped, of which 42 (26.6%) experienced signs of KTD. No statistical significant differences were observed among patients with or without KTD regarding age, gender, ethnicity and comorbidities (hypertension and diabetes). The percentage of patients with KTD was higher among those with "GG" genotype at rs1751034 of ABCC4 compared to patients without KTD [6 (14.3%) vs 4 (3.5%), p = 0.01]. No statistical significant differences were observed regarding the distribution of ABCC2 and ABCC10 SNPs. Carriers of "G" allele in homozygous status at rs1751034 of ABCC4 showed a significant association with KTD (Odds Ratio 4.67, 95% CI 1.25-17.46, p = 0.02) in bivariate analysis, but this association was lost in multivariable analysis. A significant association between bone diseases and KTD was observed (Odds Ratio 3.178, 95%CI 1.529-6.603, p = 0.002). CONCLUSIONS: According to our results ABCC4 rs1751034 could be a genetic determinant of KTD; however validation studies are needed for therapy personalization. Noteworthy, a strong association between bone disease and KTD was also observed.

Blood brain barrier impairment is associated with cerebrospinal fluid markers of neuronal damage in HIV-positive patients.

Blood brain barrier impairment occurs early in the course of infection by HIV and it may persist in a subset of patients despite effective antiretroviral treatment. We tested the hypothesis that HIV-positive patients with dysfunctional blood brain barrier may have altered biomarkers of neuronal damage. In adult HIV-positive highly active antiretroviral treatment (HAART)-treated patients (without central nervous system infections and undergoing lumbar punctures for clinical reasons) cerebrospinal fluid albumin to serum ratios (CSAR), total tau, phosphorylated tau, 1-42 beta amyloid, and neopterin were measured. In 101 adult patients, cerebrospinal fluid-to-serum albumin ratios were 4.8 (3.7-6.1) with 12 patients (11.9%) presenting age-defined impaired blood brain barrier. A significant correlation was observed between CSAR and total tau (p = 0.005), phosphorylated tau (p = 0.008), and 1-42 beta amyloid (p = 0.040). Patients with impaired blood brain barrier showed significantly higher total tau (201.6 vs. 87.3 pg/mL, p = 0.010), phosphorylated tau (35.3 vs. 32.1 ng/mL, p = 0.035), and 1-42 beta amyloid (1134 vs. 830 pg/mL, p = 0.045). Despite effective antiretroviral treatment, blood brain barrier impairment persists in some HIV-positive patients: it is associated with markers of neuronal damage and it was not associated with CSF neopterin concentrations.

Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis.

Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K-/- mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with depletion of specific neuronal populations. These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Our results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS.

Blood Brain Barrier Impairment in HIV-Positive Naïve and Effectively Treated Patients: Immune Activation Versus Astrocytosis.

Blood brain barrier (BBB) damage is a common feature in central nervous system infections by HIV and it may persist despite effective antiretroviral therapy. Astrocyte involvement has not been studied in this setting. Patients were enrolled in an ongoing prospective study and subjects with central nervous system-affecting disorders were excluded. Patients were divided into two groups: treated subjects with cerebrospinal fluid (CSF) HIV RNA <50 copies/mL (CSF-controllers) and in late-presenters CD4+ T lymphocytes <100/uL. CSF biomarkers of neuronal or astrocyte damage were measured and compared to CSF serum-to-albumin ratio. 134 patients were included; 67 subjects in each group (50 %) with similar demographic characteristics (with the exception of older age in CSF controllers). CD4 (cells/uL), plasma and CSF HIV RNA (Log10 copies/mL) were 43 (20-96), 5.6 (5.2-6) and 3.9 (3.2-4.7) in LPs and 439 (245-615), <1.69 (9 patients <2.6) and <1.69 in CSFc. BBB impairment was observed in 17 late-presenters (25.4 %) and in 9 CSF-controllers (13.4 %). CSF biomarkers were similar but for higher CSF neopterin values in late-presenters (2.3 vs. 0.6 ng/mL, p < 0.001). CSARs were associated with CSF neopterin (rho = 0.31, p = 0.03) and HIV RNA (rho = 0.24, p = 0.05) in late-presenters and with CSF tau (rho = 0.51, p < 0.001), p-tau (rho = 0.47, p < 0.001) and S100beta (rho = 0.33, p = 0.009) in CSF-controllers. In HAART-treated subjects with suppressed CSF HIV RNA, BBB altered permeability was associated with markers of neuronal damage and astrocytosis. Additional treatment targeting astrocytosis and/or viral protein production might be needed in order to reduce HIV effects in the central nervous system.

c-Jun, JNK/SAPK kinases and transcription factor NF-kappa B are selectively activated in astrocytes, but not motor neurons, in amyotrophic lateral sclerosis.

There is increasing evidence that oxidative damage plays a major role in amyotrophic lateral sclerosis (ALS), but how it contributes to motor neuron degeneration and astrocytic gliosis, two pathologic hallmarks of the disease, is unknown. A few studies have suggested that ALS motor neurons die via apoptosis and show upregulation of c-jun, an immediate early gene that is necessary for neuronal apoptosis. In order to elucidate the mechanisms of cell damage induced by oxidant stress, we have studied in ALS and control spinal cord the immunohistochemical expression of c-Jun, of JNK/SAPK, a kinase that activates c-Jun following various types of stress, and of NF-kappa B, a transcription factor that is induced by oxidant stress and has prominent neuroprotective functions. An in situ end-labeling assay was performed for detecting apoptotic cells. We show that (a) the JNK/SAPK-c-Jun pathway is dramatically overexpressed in ALS spinal cord; (b) the strongest activation occurs in astrocytes, while motor neurons show unusually low expression of the pathway; (c) increased JNK/SAPK expression in glial cells is accompanied by NF-kappa B activation, indicating the presence of a protective response to oxidant sress, which is deficient in motor neurons; (d) activation of JNK/SAPK, c-Jun and NF-kappa B is unrelated to apoptotic cell death. These results support the view that astrocytes are directly involved in the pathologic process of ALS, and might explain the selective vulnerability of motor neurons by their relative lack of antioxidant defenses.

Activation of the JNK/p38 pathway occurs in diseases characterized by tau protein pathology and is related to tau phosphorylation but not to apoptosis.

JNK and p38, two members of the MAP kinase family, are strongly induced by various stresses including oxidative stress and have been involved in regulation of apoptosis. As both kinases phosphorylate tau protein in vitro, we have investigated their immunohistochemical localization in a group of neurodegenerative diseases characterized by intracellular deposits of hyperphosphorylated tau. Cases included Alzheimer disease, Pick disease, progressive supranuclear palsy, corticobasal degeneration, Gerstmann-Sträussler-Scheinker disease-Indiana kindred, and frontotemporal dementia with parkinsonism linked to chromosome 17. In all tissue samples, strong immunoreactivity for both MAP kinases was found in the same neuronal or glial cells that contained tau-positive deposits. By double immunohistochemistry, JNK and p38 colocalized with tau in the inclusions. Analysis of apoptosis-related changes (DNA fragmentation, activated caspase-3) showed that the expression of JNK and p38 was unrelated to activation of an apoptotic cascade. Our data indicate that phospho-JNK and phospho-p38 are associated with hyperphosphorylated tau in a variety of abnormal tau inclusions, suggesting that these kinases may play a role in the development of degenerative diseases with tau pathology.

Molecular mechanisms of neuronal death in the dorsal lateral geniculate nucleus following visual cortical lesions.

We investigated the molecular mechanisms of cell death in the dorsal lateral geniculate nucleus of the rat, following suction lesion of the visual cortex at birth or in the third postnatal week, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique and immunohistochemistry for caspase-3, -7, -8, and cleaved poly(ADP-ribose) polymerase. Following lesion at birth, TUNEL-positive neurons were found in the dorsal lateral geniculate nucleus between 24 h and 3 days after lesion, with a peak on the second day. Shorter survival times (12-18 h) resulted in labeling of very few neurons in dorsal lateral geniculate nucleus and of several neurons in the perilesional cortex. Activated caspase-3 was expressed from the first to the third days after lesion, whereas cleaved poly(ADP-ribose) polymerase and activated caspase-8 were expressed on the second and third day. Activated caspase-7 was expressed mainly in pretectal nuclei. Caspase-3 activation coincided with the appearance of TUNEL-positive profiles, but decreased earlier than TUNEL. In the ipsi- and contralateral cerebral cortex, all parameters were unchanged. In animals lesioned in the third week, rare apoptotic thalamic neurons were detected as TUNEL- and activated caspase-3-positive profiles 2 days after cortical ablation, and were still present 1 week after lesion.Thus, early target ablation has dramatic effects on neonatal thalamic neurons, which die following activation of caspases 3 and 8. In contrast, cortical neurons are relatively unaffected by target deprivation. Compared with early lesions, late lesions induce a limited thalamic cell death, that persists over time.

Intestinal capillariasis (Capillaria philippinensis) acquired in Indonesia: a case report.

We report a case of intestinal capillariasis in a 32-year-old Italian man. After he made a trip to Indonesia that lasted approximately one month, he developed heartburn, abdominal pain, irregular bowel movements, headache, fatigue, weight loss, low-grade fever, and severe itching. The diagnosis was provided by the recovery of Capillaria philippinensis eggs in the stool. Treatment with oral albendazole, 200 mg twice a day for 21 days, resulted in clinical and parasitologic cure. This is the first report of C. philippinensis infection acquired in Indonesia.

Current in vitro culture systems for Pneumocystis.

Although Pneumocystis continuous culture systems have not yet been developed, efficient short-term in vitro methods allowing the production of infectious forms of Pneumocystis can now be employed. The quality of the inoculum will influence the in vitro development of P. carinii. For this reason, efficient extraction and cryopreservation techniques are considered in this section. In vitro growth and limited passage were obtained by inoculating freshly extracted parasites onto fibroblast- or epithelial-like cell monolayers cultivated in ordinary tissue culture flasks, culture plates, microcarrier beads or other culture devices. Cultures were usually maintained in an atmosphere of 5% CO2 at 35-37 degrees C. The results obtained in these different systems were surprisingly similar: the number of parasites increased about 6-10 times within the first 3-4 days post-inoculation, then remained stationary until day 7-14 and decreased rapidly. If passages were attempted, the growth decreased gradually and no growth was recorded after 2-3 passages. Proof of the in vitro Pneumocystis attachment to feeder cells has been furnished by electron microscopy. Two currently used feeder cell culture systems were selected in this subchapter. The first system is a co-culture of monolayer lung epithelial-like cells with Pneumocystis. After trypsin treatment and passage of cells with attached parasites to culture bottles containing fresh medium, 3 or more new culture bottles can be plated. A 2-4-fold increase in parasite number can be obtained but, interestingly, cultured parasites were more infectious to the nude rat than freshly extracted lung parasites. In the second system, the spinner flask culture method, Pneumocystis is cultivated on cell coated microbeads in slow stirring vessels, in order to exploit the beads' huge surface where microorganisms can transiently adhere and grow and from where they can be easily detached by simply leaving the beads to settle down. This culture system has ensured 10(8)-10(9) viable trophozoites in each harvest after 7-10 days of slow stirring incubation.

In vitro drug assays and statistical analysis.

The development of in vitro drug tests to assess the efficacy of drugs against Pneumocystis carinii has been hindered by the lack of efficient methods for continuous cultivation of the microorganism. However, different short-term culture systems have been proposed by many teams. In the present contribution an in vitro microplate drug assay and two statistical programs allowing the analysis of results are presented.

S-100beta protein is upregulated in astrocytes and motor neurons in the spinal cord of patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss and astrogliosis. We studied the immunohistochemical expression of S-100beta, a calcium-binding protein with both neurotrophic and neurotoxic activities, in the spinal cord of patients with ALS. Adjacent sections were processed with an in situ end-labeling technique for the demonstration of apoptosis-related DNA fragmentation. In controls, low expression of S-100beta was found in astrocytes but not motor neurons. Compared to controls, S-100beta was overexpressed in ALS. Most stained cells were reactive astrocytes, but a minority of motor neurons was also labeled. Neuronal labeling was unrelated to the presence of signs of atrophy/degeneration. S-100beta expression was also unrelated to neuronal or glial apoptosis. S-100beta upregulation in ALS spinal cord suggests that the protein might be involved in cellular defense mechanisms against oxidative stress.

Pneumocystis carinii diagnosis: an update.

From 1994 to date we have been using the internal transcribed spacers (ITSs) nested polymerase chain reaction (PCR) to investigate the possibility of diagnosing Pneumocystis carinii pneumonia on non-invasive samples collected from HIV-positive patients with pulmonary involvement. The objectives were: (1) to test the sensitivity, specificity and prognostic value of PCR in diagnosis and follow up of PCP; (2) to investigate the eventual occurrence and role of asymptomatic carriers of P. carinii; (3) to evaluate the prognostic significance of blood PCR positivity versus respiratory samples; (4) to verify the occurrence of exogenous infections or endogenous reactivations in cases of recurrent P. carinii pneumonia; and (5) to study the possible correlation between P. carinii genotype identified and capability of blood dissemination, prior prophylactic treatments, clinical parameters and outcome of the patients.

Pneumocystis carinii pneumonia and tuberculosis in Tanzanian patients infected with HIV.

PIP: Between August and December 1991 in Tanzania, a study to determine the prevalence of Pneumocystis carinii and of tuberculosis occurred among 83 18-38 year old HIV seropositive people living in the rural area of Malenga Makali in Iringa district. The adults had difficulty breathing, cough, fever of at least 2 weeks duration, or overt pneumonia. 3.6% of the sputum samples were confirmed positive for P. carinii. 38.5% of preparations and 13.2% of cultures tested positive for Mycobacterium tuberculosis. All these isolates were completely sensitive to standard antibiotics. 2 of the 3 patients testing positive for P. carinii also had pulmonary tuberculosis. These findings showed that sputum contains many mycobacteria. They also confirmed that TB is associated with HIV infection in several African countries and that P. carinii infection occurs less frequently than it does in Europe and the US, but occurs nevertheless. A possible explanation for the low prevalence of P. carinii infection in Africa is that more virulent infections kill AIDS patients before P. carinii pneumonia has a chance to develop. The researchers admit that their inability to use more suitable specimens obtained by bronchoalveolar lavage or transbronchial biopsy could have resulted in considerable underdiagnosis. They recommended further clinical research to determine the real importance of P. carinii in developing countries.^ieng

Biomolecular techniques to detect Pneumocystis carinii f. sp. hominis pneumonia in patients with acquired immunodeficiency syndrome.

OBJECTIVES: To verify the clinical value of two different polymerase chain reactions (PCRs) for noninvasive diagnosis and follow-up during Pneumocystis carinii f. sp. hominis pneumonia (PCP) and to analyze the P. carinii f. sp. hominis genotypes involved. METHODS: Internal transcribed spacers (ITSs) nested PCR was applied to 630 samples (bronchoalveolar lavage, sera, peripheral blood mononuclear cells, and oropharyngeal samples) from 122 patients with acquired immunodeficiency syndrome and pneumonia and 40 control samples from 20 subjects seronegative for human immunodeficiency virus. One hundred and eighty samples also were examined by mt-rRNA PCR. Bronchoalveolar lavage samples and 33 sera were analyzed by type-specific oligonucleotide hybridization. RESULTS: On bronchoalveolar lavage samples, the two PCRs consistently confirmed the morphologic diagnosis of PCP. The sensitivity of ITSs nested PCR versus mt-rRNA PCR was 57.3% versus 14.3% on sera, 32.3% versus 22. 8% on peripheral blood mononuclear cells, and 69.1% versus 48.6% on oropharyngeal samples (garglings). Both PCRs had 100% specificity. Type-specific oligonucleotide hybridization revealed in 72.2% of bronchoalveolar lavage samples a single P. carinii f. sp. hominis genotype, whereas in 27.8% co-infection with more than one strain was detected. CONCLUSION: On noninvasive samples, ITSs nested PCR was more sensitive than mt-rRNA PCR, and it confirmed the diagnosis in all patients with PCP. For each patient with PCP at least one noninvasive sample was positive for P. carinii f. sp. hominis DNA.

Madin Darby canine kidney: a new cell line for Pneumocystis carinii in vitro culture.

Pneumocystis carinii pneumonia (PCP) is a highly frequent cause of morbidity and mortality in immunocompromised subjects, particularly in HIV-infected ones. The biology of P. Carinii is poorly understood because of the lack of reliable synthetic media or adequate cell lines to grow this opportunistic pathogen in continuous culture. We reported the suitability of the MDCK (Madin Darby Canine Kidney, ATTC CCL 34) cell line to support the temporary microorganism's growth in vitro and the experimental pharmacological trials, in comparison with the HEL 299 cell line, used as reference standard.

Detection of rat Pneumocystis carinii proteinases and elastase and antipneumocystis activity of proteinase inhibitors in vitro.

Proteinases play an important role in survival of microorganisms and in pathogenicity of diseases. By using a modified SDS-gelatin-polyacrylamide gel system, proteinases of rat-P.carinii were detected as bands of proteolytic digestion after electrophoresis. P.carinii organisms obtained from dexamethasone immunosuppressed transtracheally infected rats were cultured in spinner flask suspension cultures to minimize host cell contamination. At pH 8.3, seven Pc-specific proteolytic bands were detected in three clusters of different molecular weights clearly different from host cell patterns. By using a range of pH, various preparations of organisms and both infected and uninfected culture media, proteolytic activities have been partially characterized. Elastase secretion has been assessed based on elastin digestion model. Proteinase inhibitors have been tested for their ability to inhibit P.carinii growth in HEL299 short-term monolayer cultures. Results indicate that proteolytic activities are involved in the proliferation of microorganisms since leupeptin exerted in vitro antipneumocystis activity while aprotinin enhanced P.carinii growth.