The effect of protease inhibitor-based dual antiretroviral regimens on CD4/CD8 ratio during the first year of therapy in ART-naïve patients with HIV-infection.

OBJECTIVES: To assess the effect of protease inhibitor (PI)-based dual therapy on CD4/CD8 ratio during the first year of therapy in antiretroviral therapy (ART)-naïve patients using data from randomized controlled clinical trials. METHODS: We pooled data from the GARDEL and ANDES studies, both randomized controlled clinical trials that recruited ART-naïve people living with HIV and randomly assigned them to receive PI-based dual therapy (DT) or triple therapy (TT) aiming to compare viral efficacy. We compared median CD4/CD8 ratios and the proportion of patients with CD4/CD8 ratio > 1 at 48 weeks after ART initiation in both treatment arms using the Mann-Whitney U-test and the χ2 test. We performed subgroup analysis for patients > 50 years old, with baseline CD4 counts ≤ 200 cells/µL, viral load > 100 000 HIV RNA copies/mL, and ritonavir-boosted lopinavir-based therapy. RESULTS: We analysed data from 571 patients: 292 on DT and 279 on TT. No differences were observed in CD4/CD8 ratio (0.632 vs. 0.617, P = 0.729) or in the proportion of patients with CD4/CD8 ratio > 1 (17.9% vs. 19.3%, P = 0.678) 48 weeks after ART initiation. Subgroup analysis showed no further differences. CONCLUSION: The impact of PI-based DT regimens on the CD4/CD8 ratio during the first year of treatment for ART-naïve patients is similar to that of TT.

Dual therapy with raltegravir plus a fixed dose combination of darunavir/ritonavir in people living with HIV in Argentina.

OBJECTIVE: There are generic fixed-dose combinations (FDCs) of ritonavir-boosted darunavir (DRV/r) available in Argentina. Experiences with these FDCs in dual therapy remain limited in clinical practice. We aimed to describe clinical and virologic outcomes in patients exposed to FDC DRV/r + raltegravir (RAL) 400 mg every 12 h in a real-life setting. METHODS: Retrospective analysis of electronic medical records of HIV-infected patients under FDC DRV/r + RAL in an HIV clinic in Argentina (2014-2018). Individuals were classified as "switch group" (SG, undetectable viral load [VL] with any toxicity/comorbidity) and "virologic group· (VG, detectable viremia and infection by multidrug-resistant HIV). RESULTS: Of 7,380 patients on ART, 116 (1.5%) received FDC DRV/r + RAL, being 58% in SG. Sixty percent received DRV/r 800/100 mg dose (rest, 600/100 mg). The median (IQR) age and CD4+ T-cell count were: 52 (42-58) years, and 373 cell/µL (202-642). Ninety-eight percent were ART-experienced with a median of 3 (IQR 2-5) prior treatments. Main reasons for switch (SG) were renal (57%), cardiovascular (54%) and bone (14%) comorbidities. Median exposure to DRV/r + RAL was 18 months. Among patients in SG, 98% and 96% had undetectable VL at 6 and 12 months; in the VG, 89% and 87% had undetectable VL at 6 and 12 months. No patient required suspension due to toxicity/ intolerance. CONCLUSIONS: In this cohort of mostly experienced HIV-infected patients, FDC DRV/r + RAL was effective and safe. Such therapy may be considered an option for patients with comorbid conditions and/or with multidrug-resistant HIV.

Reverse transcriptase and protease inhibitors mutational viral load in HIV infected pregnant women with transmitted drug resistance in Argentina.

OBJECTIVE: Argentina has reported high levels of transmitted drug resistance (TDR), in HIV-infected pregnant women by population sequencing. We aimed to describe, in patients with TDR, the percentage of quasispecies harboring resistance mutations (RAMs) and mutational load (ML). METHODS: Retrospective study in a cohort of 40 naïve HIV-infected pregnant women, whose pretreatment samples had been genotyped by TRUGENE (period 2008-2014). Samples were re-sequenced with Ultra-deep Sequencing and ML was calculated considering baseline HIV-1 RNA load multiplied by the frequency of quasispecies harboring RAMs. RESULTS: TDR for NNRTIs, NRTIs and PIs was 17.5% (n=7 patients), 10% (n=4), 12.5% (n=5) respectively. Predominant NNRTI RAMs were K103N (n=4; 10%) and G190A/E/S (n=3; 7.5%). For NNRTIs, 78% of RAMs were present in >93.5% of viral population and ML was >1000 copies/mL (c/mL) for 89%, with a median (IQR) of 8330 c/ml (7738-29796). The following NRTI RAMs were described (per patient: % of quasispecies, ML): T215I (99.7%, 11014 c/ml); D67G (1.28%, 502 c/mL); M41L (79.8%, 88578 c/mL) and M184I (1.02%, 173 c/mL). Most frequent PI-RAMs were I85V, M46I, I50V and L90M (n=2, 5% each). For PIs, quasispecies with RAMs were <2.3% of viral population and ML was <350 c/mL for 77.8% of them. CONCLUSIONS: NNRTI-RAMs are predominant within the viral population, usually exceeding the threshold of 1000 c/mL, indicating potential higher risk of perinatal transmission. Conversely, PI mutations appear mostly as minority variants, with potential lower risk of transmission. Among NRTI, quasispecies harboring RAMs and ML values were variable.

[Tuberculous peritonitis in HIV-infected patients]. / Peritonitis tuberculosa en pacientes infectados por el virus de la inmunodeficiencia humana.

UNLABELLED: In order to describe the clinical and laboratory findings of Mycobacterium tuberculosis peritonitis M. tuberculosis in HIV+ patients, we conducted a retrospective analysis of the medical records of HIV+ patients with isolation of M. tuberculosis from ascitic fluid (AF), assisted at Hospital Muñiz, Buenos Aires, Argentina (1996-2005). RESULTS: 21 patients were included. Median age: 33, male sex: 52%; peripheral blood CD4-T lymphocyte count (median): 85/mm3; prior history of tuberculosis: 40%; cirrhosis: 65%; enolism: 45%; HCV coinfection: 85%. The most frequent symptoms were abdominal distension (71%), fever (62%) and abdominal pain (19%). The chemical characteristics of the AF were (median): leukocyte count: 751/mm3 (mononuclear predominance: 79%), protein: 3.1 g/dl, LDH: 351 IU/l. AF samples positive for acid fast bacilli at direct microscopic examination: 14%. Infection with multidrug resistant M. tuberculosis (TB-MR): 20%. M. tuberculosis was isolated from other clinical samples in 79%. Fifteen patients received treatment for tuberculosis; in 30% of cases, it was not appropriate due to the susceptibility of the isolated strain. Overall mortality was 66.4%. CONCLUSION: high mortality was observed, which may be attributable to the high frequency of TB-MR, the level of immunosuppression and the prevalence of cirrhosis secondary to enolism and/or HCV coinfection.

Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications.

Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.

Tuberculous meningitis in HIV-infected and non-infected patients: comparison of cerebrospinal fluid findings.

We performed a retrospective comparison of cerebrospinal fluid (CSF) characteristics and drug susceptibility profile in human immunodeficiency virus (HIV) infected and non-infected patients with a diagnosis of tuberculous meningitis. HIV-infected patients had a higher frequency of non-inflammatory CSF (absence of pleocytosis) and of infection by multidrug-resistant strains of Mycobacterium tuberculosis. Protein CSF levels were lower in HIV-infected patients, while and glucose concentration was similar in both groups. Hospital mortality was significantly higher in HIV-infected patients (63.3% [64/101] vs. 17.5% [7/40]).

Resistance to HIV integrase strand transfer inhibitors in Argentina: first interim survey.

OBJECTIVE: No data on resistance to HIV integrase strand transfer inhibitors (InSTIs) in Argentina are available as access to these drugs and to integrase genotypic resistance test is limited. We aimed to evaluate the clinical profile of patients who underwent an integrase genotypic resistance test, prevalence of InSTI resistance mutations and predicted efficacy of raltegravir, elvitegravir and dolutegravir in our country. METHODS: Retrospective multicentric pilot survey from January 2011 to November 2017 of InSTI-failing patients assisted at two private and one public healthcare institutions located in Buenos Aires city, Argentina. RESULTS: Sixty seven patients were included. Patients had a median of 5 (4-7) prior treatments. All patients had InSTI-containing regimens (median exposure of 22.5 months); 94% were under raltegravir therapy and 71.9% had InSTI-resistance mutations. Predominant major mutations were N155H (35.1%), Q148H/R (15.8%) and G140A/S (14%). Considering Stanford HIVdb program, extremely low and identical activity of raltegravir and elvitegravir was described while dolutegravir remained either partially or fully active in 97.7% of patients. CONCLUSIONS: Integrase resistance test was prescribed almost exclusively in heavily pretrated raltegravir-exposed patients. The three main mutational pathways were described, with a predominance of N155H. Despite almost null susceptibility and extensive cross resistance was shown among raltegravir and elvitegravir, dolutegravir remains active in the majority of patients.

Radioimmunoassay of 2-hydroxyestrone in plasma during the estrous cycle of the rat: interrelationships with estradiol, progesterone, and the gonadotropins.

A RIA for 2-hydroxyestrone (2-OHE1) in rat plasma has been developed. The assay employs an antiserum that is specific for catechol estrogens. Specificity is further ensured by purification of plasma extracts on Sephadex LH-20 columns before RIA. Blood was collected at 0 C in the presence of ascorbic acid to prevent oxidation. Under these conditions, the conversion of 2-OHE1 to methylated derivatives was found to be negligible. Plasma 2-OHE1, LH, FSH, PRL, estradiol, and progesterone were measured at 3-h intervals throughout the 4-day estrous cycle of the rat. The 2-OHE1 concentration varied from undetectable to 11 pg/ml plasma. No clearly defined relationship with the other hormones analyzed was observed. Thus, it is unlikely that changes in circulating 2-OHE1 levels are involved in the regulation of the gonadotropin surge and ovulation.

Chromatographic enzyme immunoassay for T-2 toxin.

Both the active ester and maleimide moieties of the cross-linking reagent, N-[(gamma-maleimidobutyryl)oxy]succinimide (GMBS), were found to react with the primary amino groups on ribonuclease (RNase). This largely inactivated RNase towards a polymeric (but not monomeric) substrate. Citraconylating the RNase first, so that essentially only a single primary amino group remained to react with GMBS, overcame this problem. The subsequent maleimido-citraconyl-RNase was used to prepare a 1:1.1 M conjugate of anti-T-2 toxin Fab' and RNase (Fab'-RNase) in a 76% yield. The conjugate was used to detect as little as 0.1 microgram of T-2 toxin based on the ability of T-2 toxin to specifically displace Fab'-RNase complexed to a T-2 agarose affinity gel.

Urinary catechol estrogens in cycles stimulated by human menopausal gonadotropin.

Catechol estrogens, estrogen metabolites of potential physiologic significance, were measured in infertile women undergoing ovulation induction with human menopausal gonadotropins. Urinary 2-hydroxyestrone (2-OH-E1) specimens were obtained from 12 women in one or more stimulated cycles. The actual time for the administration of human chorionic gonadotropin to induce ovulation was based on serial plasma estradiol (E2) specimens. A significant correlation between plasma E2 and urinary 2-OH-E1 was demonstrated, similar but more pronounced than that seen in normal cycling women. This confirms previous work that showed that 2-OH-E1 is the major urinary estrogen metabolite in the nonpregnant state and further suggests that urinary catechol estrogens are a useful index of ovarian function.

A radioimmunoassay method for urinary catechol estrogens.

A radioimmunoassay method for urinary catechol estrogens is described; The specific nature of the antisera allows direct analyses of acid hydrolyzed urine. A LH-20 Sephadex column chromatography can be employed for individual determinations of 2-hydroxyestrone and 2-hydroxyestradiol. The excretion of catechol estrogens during menstrual cycles ranged from 14.48 to 50.15 microgram per 24 hours, whereas, during the last trimester of pregnancies, the values ranged from 129.30 to 1758. 20 microgram per 24 hours.

The absence of a catechol estrogen effect on blood pressure in the male rat.

Since catechol estrogens are potent competitive inhibitors of catechol-O-methyl transferase (COMT), it has been suggested that they may prolong the half-life of catecholamines which in turn can cause hypertension. Thus, experiments were carried out to study the effect of catechol estrogens on blood pressure in the male rat following chronic administration. Results demonstrate that 2-hydroxyesterone (2,3-dihydroxyestra-1,3,5(10)-trien-17-one) and 2-hydroxy-estradiol (estra-1,3,5(10)-triene-2,3,17 beta-triol) even when administered in high doses do not alter blood pressure.

"Chitin Leash": a polysaccharide heterobifunctional cross-linking agent which can be cleaved by lysozyme.

6-O-[(2-Hydroxyethyl)poly(2-oxyethyl)]chitosan ("glycolchitosan") was oxidatively cleaved with nitrous acid and then partly acetylated with acetic anhydride, reacted with bromoacetyl-N-hydroxysuccinimide, and reacted further with acetic anhydride. Conditions were selected, including fractionation by size-exclusion chromatography, so that the resulting "Chitin Leash" had an estimated, average molecular weight of 10,000 (dextran standards), corresponding to a length of approximately 40 sugar residues. It possessed 0.9 terminal aldehyde and 2.6 random (presumably) side-chain bromoacetyl reactive groups per chain (average values). As a model system, the Chitin Leash was used to crosslink staphylococcal nuclease (SNase) to ribonuclease A (RNase) with retention of 75 and 78%, respectively, of the starting enzyme activities. For this coupling, the Nase was first converted to a sulfhydryl SNase derivative which retained 74% of the activity of starting enzyme. The yields in this synthesis were: 13% Chitin Leash from glycolchitosan, 24% Chitin Leash-RNase from Chitin Leash and 45% SNase-Chitin Leash-RNase from the latter conjugate. The ratio of SNase to RNase in this conjugate was 1.0:0.94. In a second preparation, in which [14C]acetic anhydride was used, a longer reaction time was employed for the coupling of Chitin Leash to RNase. This gave a 1.0:1.8:0.95 molar ratio of Nase: [14C]Chitin Leash: RNase, revealing multiple attachment of the [14C]Chitin Leash to RNase. The activity of the RNase in the final conjugate was 20%. The latter conjugate was approximately 70% hydrolyzed by diaminooctyl-succinyl-lysozyme, disconnecting the two enzymes while not affecting their activities.

Staphylococcal nuclease high-performance liquid chromatographic characterization of diaminooctane-modified DNA and its biotin and fluorescein derivatives.

DNA was subjected to bisulfite-catalyzed transamination at the N4 sites of its cytosine residues with 1,8-diaminooctane (DAO). The product, DNA-DAO, was non-specifically degraded with a cloned staphylococcal nuclease (Nase). The products from the Nase digestion were determined by high-performance liquid chromatography (HPLC) to define the extent of reaction with DAO. Mostly, nucleoside 3'-monophosphates were obtained, along with four Nase-resistant dinucleotides: TpdGp, dApdGp, TpdCp-DAO and dApdCp-DAO. The addition of spleen phosphodiesterase II gave a faster hydrolysis and left no dinucleotides. A mixture of Nase, snake venom phosphodiesterase I and alkaline phosphatase gave a fast hydrolysis as well but two dinucleotides, apparently TpdC-DAO and dApdC-DAO, persisted. Further modification of the diaminooctyl side chains with fluorescein isothiocyanate or biotin N-hydroxysuccinimide ester was similarly investigated. Interestingly, derivatization of the DAO side chain with biotin eliminates the resistance of TpdCp-DAO and dApdCp-DAO to Nase digestion. This work provides some guidelines for using Nase, alone or with other nucleases, along with HPLC, for characterizing alkyldiamine DNA products, and should be similarly useful for studying other modifications of DNA.