Alessandra Romito

Overexpression of Fas antigen on T cells in advanced HIV-1 infection: Differential ligation constantly induces apoptosis

Objectives:To investigate Fas in peripheral lymphocytes from HIV-1-positive patients at different disease stages with respect to the extent of apoptosis. Design:The study included analysis of Fas involvement in T-cell apoptosis observed during HIV-1 infection. Because ligation of Fas can result in costimulation of proliferation or the induction of apoptosis in uninfected cells, we evaluated the effect on T cells of Fas activation by monoclonal antibodies (MAb) of different specificity from both UB2 and CH11 clones and activation by the Fas ligand (Fas-L). Methods:Fas was measured by FACS in peripheral blood and in phytohaemagglutinin (PHA)-driven cultures derived from 59 HIV-1-positive individuals with different Centers for Disease Control and Prevention stages. The percentage of apoptotic cells was detected by propidium iodide cell staining. The effect of Fas ligation was assessed in peripheral T cells from patients and healthy controls by a proliferative test measuring the 3H-thymidine uptake. Results:FACS analysis revealed that Fas was predominantly expressed in advanced disease, although it was promptly exposed in PHA cultures from asymptomatic individuals. In several instances, Fas overexpression was associated with substantial subdiploid DNA content in cells from severely lymphopenic patients. The proliferative assay showed a significant inhibition of 3H-thymidine uptake in T cells from all patients following Fas ligation by the immunoglobulin (Ig) G1 MAb from the UB2 clone. This was in contrast to the apparent cell activation detected in controls and the weak suppression observed in Fas-positive cell lines. In addition, the IgM anti-Fas and recombinant Fas-L concentrations inducing a moderate inhibition of fresh T cells from controls strongly depressed the proliferative rate of cells from patients. Conclusions:Our data suggest that Fas overexpression parallels the progression of the disease and that the increased susceptibility of T cells from HIV-1-infected individuals to undergo apoptosis may include a Fas pathway. Functionally exhausted T cells in advanced HIV-1 infection are primed to apoptosis because of their high sensitivity to Fas stimulation even using the IgG1 MAb, which is unreactive to the death domain of Fas. This suggests that the increased sensitivity of Fas is apparently unrelated to its trimeric ligation and supports the hypothesis that Fas pathway plays a role in increasing the lymphocyte apoptosis during the disease.

Long-term therapy with recombinant human erythropoietin (rHu-EPO) in progressing multiple myeloma

Recombinant human erythropoietin (rHu-EPO) is an effective growth factor for erythroid progenitor cells in anemia provoked by several conditions, including bone marrow tumors such as multiple myeloma (MM). We studied a group of 54 patients with MM undergoing second-induction chemotherapy. Thirty of them were randomly assigned to receive rHu-EPO at an initial dosage of 150 units/kg body weight three times a week, increased to 300 units/kg from the sixth week to the end of the 24-week study. Hemoglobin (Hb) levels increased in 77.7% of these patients by the eighth week. In addition, five transfusion-dependent patients in treatment with the VMCP protocol completed the trial without requiring blood supplement after the third month, whereas seven control patients required frequent supplements. Monthly assessment of hematologic parameters demonstrated the ability of rHu-EPO to increase reticulocyte counts, whereas five patients became resistant to the second-induction chemotherapy in apparent concurrence with their rHu-EPO therapy. The response to rHu-EPO in four of the five MM patients receiving cytotoxic protocols combined with α-interferon (α-IFN) included an increase of serum IgM after the third month. This effect was not demonstrable in any other group, including three rHu-EPO-untreated patients undergoing α-IFN + VMCP combined therapy, as well as rHu-EPO-treated patients not receiving a-IFN. Our data suggest that α-IFN plus rHu-EPO treatment in MM patients is effective in restoring normal B cell function. These results may reflect in vivo the modulation of normal human B cells and lymphoblasts by rHu-EPO observed in vitro.

Cerebrospinal fluid biomarkers in patients with plasma HIV RNA below 20 copies/mL

Low level HIV‐1 CSF replication (CsfLLV) is often found even in patients with controlled plasma viraemia. The clinical consequences of such finding are uncertain; however, both symptomatic neurological disturbances and neurocognitive disorders may arise in the context of CSF‐escape. Two reports suggested that low level replication in the CSF may be associated with increased CSF neopterine although the impact on other markers of neuroinflammation/damage is currently unknown.

Clinical and neuropathological findings in Hashimoto's encephalopathy: a case report

Hashimoto’s encephalopathy (HE) is a controversial autoimmune condition of the central nervous system (CNS) characterized by focal and/or diffuse symptoms concomitant with raised levels of anti-thyroid antibodies, in particular anti-thyroperoxidase (TPO) and/or anti-thyroglobulin (TG) antibodies [1]. Hereby, we provide the full clinical and neuropathological description of an HE patient with a rapid and fatal course. A 67-year-old man with autoimmune hypothyroidism taking L-thyroxine 100 lg/day came to our observation because of 1-month history of apathy and speech difficulties. At admission in the neurological ward, fluent aphasia, disorientation, and spontaneous diffuse myoclonic jerks were present. Cranial CT and brain MRI were unremarkable. EEG evidenced rare diffuse theta-delta bursts. On laboratory investigations, we detected only a markedly raised titer of serum anti-TPO antibodies (3,790 UI/ml). CSF analysis disclosed very high protein concentration (126 mg/dL) with moderate alteration of blood–brain barrier permeability. Virological analyses were all negative. Total tau CSF concentration was normal, whereas the 14-33 protein was present in traces. To rule out a neurological paraneoplastic syndrome, the patient’s serum was analyzed by an indirect immunofluorescence assay on monkey cerebellum slides. We evidenced a Tr-like pattern, characterized by a prominent staining of cytoplasm and dendrites of Purkinje cells, but with no positive dots in the molecular layer (Fig. 1). No paraneoplastic antibody specificity (Hu, Yo, Ri, amphiphysin, Ma2, Ta) was detected by dot blot. Serum anti-VGKC and anti-NMDA-R antibodies were absent. A total body contrast-enhanced CT ruled out occult neoplasms. Given the subacute encephalopathy picture with no evident causes and the very raised serum titer of anti-TPO antibodies, a presumptive diagnosis of HE was made. A high-dose systemic methylprednisolone pulse (1,000 mg/ day) was started. After 5 days from steroid initiation, the patient was intubated and transferred to the intensive care unit (ICU) because of a refractory convulsive status epilepticus. Thiopentone was administered to get a suppression-burst pattern on EEG recordings. After 1 week the patient’s clinical conditions markedly ameliorated and no seizure relapses were observed. Therefore, he was readmitted to our neurologic ward. In the following 5 days, diffuse myoclonic jerks progressively increased, leading to convulsive status epilepticus. The patient entered again the ICU to employ intravenous thiopentone. A new brain MRI study evidenced diffuse hyperintensity of cortical white matter of parietal lobes in T2 and FLAIR scans with no contrast enhancement in T1 sequences. MRI signs of bilateral hippocampal sclerosis were also present. The patient’s neurologic status deteriorated in spite of a 10-day systemic methylprednisolone (1,000 mg/day) and 5-day intravenous human immunoglobulin pulse (400 mg/kg/ day). Exitus occurred from a multiorgan failure syndrome after 3 weeks. The autopsy procedure was limited to the intracranial content, since the remote suspect of a prion disease. On gross inspection, cortical gyri were slightly flattened. On microscopic examination, areas of neuropil vacuolation with congestion and swollen endothelial cells D. Imperiale (&) R. Testi A. Romito S. Taraglio Centro Regionale Diagnosi ed Osservazione Malattie Prioniche DOMP, ASL To2, Via Cibrario 72, 10144 Turin, Italy e-mail: daniele.imperiale@aslto2.piemonte.it

Cerebrospinal fluid viral load and neopterin in HIV-positive patients with undetectable viraemia.

BACKGROUND Cerebrospinal fluid (CSF) HIV RNA is commonly used as a marker of compartmental antiviral activity in HIV-positive patients. Undetectable CSF HIV RNA levels have been associated with low CSF neopterin levels and better neurocognitive performances. The aim of this study was to analyse the prevalence and predictors of non-detectable CSF HIV RNA using a commercial assay. METHODS In adult HIV-positive HAART-treated patients with confirmed plasma HIV RNA <50 copies/ml, CSF HIV RNA (with Roche Amplicor Assay) and neopterin were measured. RESULTS 112 adult patients were included. Plasma and CSF HIV RNA were non-detectable (target not detected [TND]) in 29 (25.9%) and 36 (32.1%) patients, respectively. CSF TND was observed more frequently in patients with plasma TND (P=0.005, OR=3.87). CSF neopterin levels were associated with age (rho =0.333, P=0.002) and current (rho= -0.272, P=0.015) and nadir (rho =-0.240, P=0.038) CD4+ T-lymphocytes; the lowest CSF neopterin concentration was observed in patients with CSF TND versus other viral load strata (0.62 mg/dl versus 0.78 mg/dl; P=0.048). CONCLUSIONS Efficaciously treated HIV-positive patients with detectable plasma HIV RNA might imperfectly control CSF viral replication. Prospective studies addressing the management and neurocognitive consequences of CSF low-level viraemia are warranted.

Symptomatic cerebrospinal fluid HIV-1 escape with no resistance-associated mutations following low-level plasma viremia

The majority of neurologically symptomatic cerebrospinal fluid HIV-1 escape cases are connected with resistance-associated mutations and potentially explained by low cerebrospinal fluid antiretroviral concentrations. However, there are still significant knowledge gaps regarding the physiopathology and long-term management of neurosymptomatic viral escape. We report a case of Parkinson-like syndrome following cerebrospinal fluid HIV-1 escape in a 40-year-old female patient with an history of persistent low-level plasma viremia under treatment. No resistance-associated mutations, high viral diversity (env deep sequencing), adequate pharmacokinetics, atypical CD3-CD14-CD4+CD5-CD2-/+CD7-/+ lymphocytes, low-level Epstein-Barr virus replication, and white matter autoimmune reactivity were observed in the cerebrospinal fluid. Antiretroviral regimen modification led to rapid clinical and radiological improvements. This case may increase the current uncertain knowledge on the origin of cerebrospinal fluid HIV-1 and illustrates the consequences of uncontrolled compartmental viral replication; it also highlights the relevance and persistence of immune activation and the possibility of various detrimental mechanisms underlying neurosymptomatic viral escape.