Endoscopic transorbital approach for recurrent spheno-orbital meningiomas: single center case series.

Endoscopic transorbital approaches (ETOAs) are finding wide application for skull base lesions, particularly for spheno-orbital meningiomas (SOMs). These tumors have high recurrence rates, and second surgery can often represent a challenge. In this study we analyze our experience of management of recurrent SOMs through a slightly modified eyelid crease approach. Between May 2016 and September 2023, in the Department of Neurosurgery of Fondazione IRCCS Policlinico San Matteo (Pavia, Italy), five consecutive recurrent SOMs have been treated using an endoscopic transorbital approach. Demographic data, preoperatory deficits, lesions characteristics, histology, grade of resection, eventual adjuvant treatments, complications, outcome in terms of symptoms improvement and cosmesis, and hospitalization are described. One patient maintained a right lateral rectus muscle palsy that was already present in the preoperatory, no cerebrospinal fluid (CSF) leaks were reported. All patients had postoperative periorbital edema, but no other systemic complication was found. All patients had proptosis improvement, two had visual acuity improvement, and best cosmetic outcome was obtained in all cases. Hospitalization varied between 4 and 6 days. ETOAs in the management of recurrent SOMs are safe and have good outcome. Right selection of patients is mandatory, but when feasible, endoscopic surgery can allow a virgin route to a previously operated tumor, guaranteeing a good strategic option.

Factors Predicting Patient's Allocation to Short- and Long-Term Therapeutic Community Treatments in the Italian VOECT Cohort Study.

The Evaluation of Therapeutic Community Treatments and Outcomes (VOECT) study was conducted in 131 Italian Therapeutic Communities (TCs) in 2008/2009. All of the patients entering residential treatment for drug or alcohol dependence were invited to participate. Data regarding patient socio-demographic characteristics, drug and alcohol consumption, health and psychopathological status, prior treatments and outcomes, and their motivation score were collected upon enrolment onto the study. The aim of this work was to identify the factors associated with allocation to short- versus long-term programmes in drug or alcohol dependent patients entering TCs in Italy. Of the 2470 patients included in the analysis, 30.8% were allocated to short-term treatment and 69.2% to long-term treatment. Several factors were significantly associated with the allocation to short- and long-term treatments: unstable living conditions; entering the TC when not detoxified; a high Symptom Checklist-90 somatization score; prior cessation episodes; previous in-patient detoxification treatments; psychosocial treatments; entering the TC by oneself; and a low motivation score.

Goat casein genotypes are associated with milk production traits in the Sarda breed.

The aim of the current work was to analyze, in the Sarda breed goat, genetic polymorphism within the casein genes and to assess their influence on milk traits. Genetic variants at the CSN1S1, CSN2, CSN1S2 and CSN3 gene loci were investigated using PCR-based methods, cloning and sequencing. Strong alleles prevailed at the CSN1S1 gene locus and defective alleles also were revealed. Null alleles were evidenced at each calcium-sensitive gene locus. At the CSN3 gene locus, we observed a prevalence of the CSN3 A and B alleles; the occurrence of rare alleles such as CSN3 B'', C, C', D, E and M; and the CSN3 S allele (GenBank KF644565) described here for the first time in Capra hircus. Statistical analysis showed that all genes, except CSN3, significantly influenced milk traits. The CSN1S1 BB and AB genotypes were associated with the highest percentages of protein (4.41 and 4.40 respectively) and fat (5.26 and 5.34 respectively) (P < 0.001). A relevant finding was that CSN2 and CSN1S2 genotypes affected milk protein content and yield. The polymorphism of the CSN2 gene affected milk protein percentage with the highest values recorded in the CSN2 AA goats (4.35, at P < 0.001). The CSN1S2 AC goats provided the highest fat (51.02 g/day) and protein (41.42 g/day) (P < 0.01) production. This information can be incorporated into selection schemes for the Sarda breed goat.

The weekend factor: the effect on healthcare quality. A medico-legal, retrospective study.

Background: In the healthcare system, in the last 30 years, the prognostically negative value of the so-called Weekend Effect (WE) has been internationally recognized. The WE is regarded as the increased risk a patient might incur when hospitalized during non-working days, of enduring severe complications in comparison to the same hospitalization that occur on working days. The aim of this study was to retrospectively verify whether, once a mistake was made during weekends or on holidays, in comparison to a mistake occurred on workdays, it subsequently implied a higher risk of complications, death included, in a statistical and medico-legal way. Methods: Three different evaluators independently examined a total of 378 medico-legal cases over a more than 20-year period. Eventual medical actions and omissions were labelled as 'mistake' when the AJ claimed that at least one occurred; 'alleged mistake' included the cases where the EW's report disagreed with the AJ's one; finally, 'no mistake' when both the AJ and the EW agreed in their evaluations. During weekends there is a higher risk that a mistake occurs (OR=3.3, 95% CI=1.6;7.4; p-value<0.001) compared to weekdays. When death occurs, delayed diagnosis is the main cause (p=0.02), whereas a damaging action is more frequently claimed in general. Conclusions: We verified as actual the impact of the WE on patients' outcome from a medico-legal point of view. The implications for an improvement of the several settings of the Italian NHS are various, and many are the consequences in the healthcare management.

Association between rainfall and Escherichia coli in live bivalve molluscs harvested in Sardinia, Italy.

Rainfall is generally accepted as one of the most important factors associated with an increased level of E. coli in bivalve molluscs. Performing microbiological risk assessment is relevant to official control authorities to determine the sanitary status of harvesting areas and, therefore, develop monitoring strategies and identify management practices that could be used to improve the quality and safety of the final product. The present study aimed to investigate the impact of rainfall on the content of E. coli in bivalve molluscs farmed in Sardinia (Italy). Enumeration of E. coli was performed according to the Most Probable Number (MPN) method (ISO 16649-3) on 1,920 bivalve samples collected from 7 regional counties between 2018 and 2020. Bivalve molluscs samples included 955 mussels (Mytilus galloprovincialis), 500 oysters (Crassostrea gigas), 325 clams (Ruditapes decussatus), 94 warty venus (Venus verrucosa), and 46 lagoon cockles (Cerastoderma glaucum). Rainfall data were obtained by the Department of Meteorology of the ARPA Sardegna. For each sampling site, GPS coordinates were used to identify gauge stations within catchment areas. Cumulative rain (mm) was recorded 1, 3, 5, 7, and 15 days before sampling, among which the 7-day cumulative rain was the strongest predictor of E. coli counts. Several thresholds of 7-day cumulative rain (from <10 mm up to >300 mm) before sampling were used to estimate the chances of a non-compliant sample (E. coli levels above the limit for sanitary class A; 230 MPN/100 g). The 7-day cumulative rain was positively associated with the chances of non-compliance. When the 7-day cumulative rain before sampling was >300 mm, 80.5 % of the samples were non-compliant, and the odds of a non-compliant sample were 23.6 times higher, as compared to samples harvested when the 7-day cumulative rainfall was <10 mm. Precipitation data could be a useful tool for interpreting anomalous results from official control authorities and reduce the costs that originate from closure of production areas.

SARS-CoV-2 systemic infection in a kidney transplant recipient: sequence analysis in clinical specimens.

OBJECTIVE: Herein we report clinical and virological data in a patient with COVID-19 infection and a prior history of kidney transplantation who had a good clinical recovery despite systemic infection. PATIENTS AND METHODS: Reverse transcriptase quantitative PCR analysis for the RdRp, N and E target genes detected viral RNA in different types of biological specimens. Whole viral genome sequences were obtained and analyzed from respiratory tract, feces and blood. RESULTS: Viral sequences showed high (~99.9%) homology with the Wuhan seafood market pneumonia virus. Phylogenetic analysis assigned of the SARS-CoV-2 strains to clade G. A rare variant in the orf1ab gene was present in both sequences, while a missense variant was detected only in viral RNA from stool. CONCLUSIONS: The evolution of the COVID-19 systemic infection in the patient presented here was favorable to the hypothesis that immunosuppressive therapy in organ transplant recipients might be involved in viral dissemination. A missense mutation was present in only one specimen from the same patient implying the occurrence of a mutational event in viral RNA, which is suggestive for the presence of an active virus, even though viral isolation is necessary to demonstrate infectivity.

A role for TGF-beta in oligodendrocyte differentiation.

Oligodendrocyte-type-2 astrocyte (O-2A) glial progenitor cells undergo a limited number of mitotic divisions in response to PDGF before differentiating into oligodendrocytes, the myelin-forming cell of the CNS. We examined the mechanism limiting O-2A proliferation, and demonstrate that these cells secrete an inhibitor of cell proliferation that can be neutralized with antibodies to TGF-beta. O-2A cells also secrete an inhibitory activity that cannot be neutralized with TGF-beta antibodies. O-2A progenitor cultures express TGF-beta 1 isoform and its transcript, while oligodendrocyte cultures express TGF-beta 1, beta-2, and beta-3 isoforms. Both recombinant TGF-beta 1 and O-2A conditioned medium inhibit the proliferation of O-2A progenitor cells cultured in the presence of PDGF, and this inhibition can be partially neutralized with polyclonal TGF-beta antibodies. Thus, TGF-beta produced by O-2A cells may limit PDGF-driven mitosis and promote oligodendrocyte development. TGF-beta is a less potent inhibitor of O-2A proliferation when these cells are cultured in the presence of bFGF, suggesting that bFGF interferes with TGF-beta signaling. Thus, the production of TGF-beta by cells in the O-2A lineage may account for the distinct effects of PDGF and bFGF on O-2A progenitor cell proliferation. Moreover, our results suggest that TGF-beta may be an important mediator of oligodendrocyte differentiation.

Extracorporeal portal vein oxygenation improves outcome of acute liver failure in swine.

BACKGROUND: Portal vein arterialization (PVA) has shown efficacy to treat acute liver failure (ALF) in preclinical studies. The next step is to perform large animal studies that propose a clinically acceptable method of PVA. In this study, we assessed the efficacy of PVA using an extracorporeal device to treat 2 ALF models in swine. MATERIALS AND METHODS: The 2 ALF swine models were carbon tetrachloride toxic ALF and subtotal hepatectomy using 8 animals per group. PVA was performed with an extracorporeal device that may be suitable for future clinical studies. Arterial blood was drawn from the iliac artery and delivered into the portal vein for a 6-hour treatment. We analyzed biochemical, blood gas, and histological parameters as well as 1-week survival rates. RESULTS: In both models, ALF was successfully achieved. Control group animals deteriorated biochemically, dropping their prothrombin times and increasing the liver enzymes. In contrast, treated animals improved with a survival rate of 75% at 7 days compared with 0% for the former group. CONCLUSIONS: PVA using an extracorporeal device was feasible and effective to treat both toxic and resective ALF in swine.

Transcriptional activation of the integrated chromatin-associated human immunodeficiency virus type 1 promoter.

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression involves a complex interplay between cellular transcription factors, chromatin-associated proviral DNA, and the virus-encoded transactivator protein, Tat. Here we show that Tat transactivates the integrated HIV-1 long terminal repeat (LTR), even in the absence of detectable basal promoter activity, and this transcriptional activation is accompanied by chromatin remodeling downstream of the transcription initiation site, as monitored by increased accessibility to restriction endonucleases. However, with an integrated promoter lacking both Sp1 and NF-kappaB sites, Tat was unable to either activate transcription or induce changes in chromatin structure even when it was tethered to the HIV-1 core promoter upstream of the TATA box. Tat responsiveness was observed only when Sp1 or NF-kappaB was bound to the promoter, implying that Tat functions subsequent to the formation of a specific transcription initiation complex. Unlike Tat, NF-kappaB failed to stimulate the integrated transcriptionally silent HIV-1 promoter. Histone acetylation renders the inactive HIV-1 LTR responsive to NF-kappaB, indicating that a suppressive chromatin structure must be remodeled prior to transcriptional activation by NF-kappaB. Taken together, these results suggest that Sp1 and NF-kappaB are required for the assembly of transcriptional complexes on the integrated viral promoter exhibiting a continuum of basal activities, all of which are fully responsive to Tat.

Interaction of the human T-cell lymphotropic virus type 1 tax transactivator with transcription factor IIA.

The Tax protein of human T-cell lymphotropic virus type 1 (HTLV-1) is a 40-kDa transcriptional activator which is critical for HTLV-1 gene regulation and virus-induced cellular transformation. Tax is localized to the DNA through its interaction with the site-specific activators cyclic AMP-responsive element-binding protein, NF-kappaB, and serum response factor. It has been suggested that the recruitment of Tax to the DNA positions Tax for interaction with the basal transcriptional machinery. On the basis of several independent assays, we now report a physical and functional interaction between Tax and the transcription factor, TFIIA. First, Tax was found to interact with the 35-kDa (alpha) subunit of TFIIA in the yeast two-hybrid interaction system. Importantly, two previously characterized mutants with point mutations in Tax, M32 (Y196A, K197S) and M41 (H287A, P288S), which were shown to be defective in Tax-activated transcription were unable to interact with TFIIA in this assay. Second, a glutathione-S-transferase (GST) affinity-binding assay showed that the interaction of holo-TFIIA with GST-Tax was 20-fold higher than that observed with either the GST-Tax M32 activation mutant or the GST control. Third, a coimmunoprecipitation assay showed that in HTLV-1-infected human T lymphocytes, Tax and TFIIA were associated. Finally, TFIIA facilitates Tax transactivation in vitro and in vivo. In vitro transcription studies showed reduced levels of Tax-activated transcription in cell extracts depleted of TFIIA. In addition, transfection of human T lymphocytes with TFIIA expression vectors enhanced Tax-activated transcription of an HTLV-1 long terminal repeat-chloramphenicol acetyltransferase reporter construct. Our study suggests that the interaction of Tax with the transcription factor TFIIA may play a role in Tax-mediated transcriptional activation.

Zac1 (Lot1), a potential tumor suppressor gene, and the gene for epsilon-sarcoglycan are maternally imprinted genes: identification by a subtractive screen of novel uniparental fibroblast lines.

Imprinted genes are expressed from one allele according to their parent of origin, and many are essential to mammalian embryogenesis. Here we show that the epsilon-sarcoglycan gene (Sgce) and Zac1 (Lot1) are both paternally expressed imprinted genes. They were identified in a subtractive screen for imprinted genes using a cDNA library made from novel parthenogenetic and wild-type fibroblast lines. Sgce is a component of the dystrophin-sarcoglycan complex, Zac1 is a nuclear protein inducing growth arrest and/or apoptosis, and Zac1 is a potential tumor suppressor gene. Sgce and Zac1 are expressed predominantly from their paternal alleles in all adult mouse tissues, except that Zac1 is biallelic in the liver and Sgce is weakly expressed from the maternal allele in the brain. Sgce and Zac1 are broadly expressed in embryos, with Zac1 being highly expressed in the liver primordium, the umbilical region, and the neural tube. Sgce, however, is strongly expressed in the allantoic region on day 9.5 but becomes more widely expressed throughout the embryo by day 11.5. Sgce is located at the proximal end of mouse chromosome 6 and is a candidate gene for embryonic lethality associated with uniparental maternal inheritance of this region. Zac1 maps to the proximal region of chromosome 10, identifying a new imprinted locus in the mouse, homologous with human chromosome 6q24-q25. In humans, unipaternal disomy for this region is associated with fetal growth retardation and transient neonatal diabetes mellitus. In addition, loss of expression of ZAC has been described for a number of breast and ovarian carcinomas, suggesting that ZAC is a potential tumor suppressor gene.

Portal vein arterialization in hepatobiliary surgery and liver transplantation.

We reviewed the literature reports and our personal experience on partial portal vein arterialization (PPVA) to prevent and treat acute liver failure (ALF) following major hepatobiliary surgery or another etiology. Experimental studies in rats have assessed the efficacy of PPVA in treatment of ALF induced by extended resections in normal or fatty livers or in toxic carbon-tetrachloride damage. The treated groups showed greater survival and faster recovery of liver function. Among 11 clinical cases reported in the literature, PPVA was performed in four cases to prevent and in seven cases to treat ALF. Eight patients survived, showing rapid recovery of liver function and resolution of the clinical condition. This relatively simple procedure has shown itself able to promote liver regeneration. The PPVA procedure has shown itself to be safe and simple as well as to offer a promising approach to the failing liver.

Protective effect of an inhibitor of interleukin-8 (meraxin) from ischemia and reperfusion injury in a rat model of kidney transplantation.

INTRODUCTION: Since the ischemia and reperfusion injury is one of the main causes of delayed graft function after transplantation, research efforts have focused on studying the molecules involved in this inflammatory process. The chemokine interleukin-8 (IL-8) seems to be the main one responsible through a chemoattractive action toward neutropils. Therefore, one of the strategies adopted to prevent this process is blocking the binding between IL-8 and its receptors. The aim of our study was to test the effect of meraxin, a new derivative from repertaxin, to protect the renal graft from ischemia and reperfusion injury. MATERIALS AND METHODS: Eighty male syngenic rats were divided into four groups. The control group underwent only kidney transplantation, while the other groups were treated with meraxin at various dosages 2 hours before graft reperfusion. Blood and histological samples were taken at sacrifice 24 hours after transplantation. RESULTS: Creatinine was significantly lower in the group treated with the high dosage of meraxin. Histological observation of the grafted tissue showed instead only a mild and not significant neutrophilic infiltration, equal in each group. CONCLUSIONS: Graft function was improved by the administration of meraxin at high dosage, but this effect did not seem to be connected to a reduction in inflammatory infiltration in the parechymal tissue. Maybe the cause is in the mechanisms of clotting activation, due to alteration of adhesion molecules and endothelial cells.

[Resident psychiatrists in Amsterdam recognize the value of personal therapy]. / De waardering van leertherapie door psychiaters in opleiding in Amsterdam.

BACKGROUND: Resident psychiatrists in the Netherlands, unlike their counterparts in other countries, are obliged to undergo 50 hours of personal psychotherapy. In 1994 Trijsburg et al. published the results of a questionnaire that had been completed by psychiatrists, psychologists and other persons training to become psychotherapists. AIM: To fnd out how resident psychiatrists in Amsterdam in 2003 characterised and rated the therapy module in their course. METHOD: Resident psychiatrists in Amsterdam were asked to complete a shortened version of the 1994 questionnaire. The Utrecht Burn-out Scale was added. RESULTS: Personal therapy was greatly appreciated, but the psychotherapists were more convinced of the positive effects than were the residents. Hardly anyone referred to any negative aspects. The residents did not consider personal therapy to be an essential element in their course. Two-thirds of the residents in Amsterdam were female, a large increase compared to the male-female ratio in 1994. The workload of the residents was in accordance with that of other workers in the health care sector. CONCLUSION: Since resident psychiatrists in Amsterdam have a positive attitude to compulsory personal therapy, there seems to be no compelling reasonfor abolishing this module--even though the beneficial effects of this therapy have never been demonstrated.

A PC-1 amino acid variant (K121Q) is associated with faster progression of renal disease in patients with type 1 diabetes and albuminuria.

Insulin resistance characterizes type 1 diabetes in patients with albuminuria. A PC-1 glycoprotein amino acid variant, K121Q, is associated with insulin resistance. We examined the impact of the PC-1 K121Q variant on the rate of decline of the glomerular filtration rate (GFR) by creatinine clearance derived from the Cockroft-Gault formula in 77 type 1 diabetic patients with albuminuria who were followed for an average of 6.5 years (range 2.5-15). Patients carrying the Q allele (n = 22; 20 with KQ and 2 with QQ genotypes) had a faster GFR decline than those patients with the KK genotype (n = 55) (median 7.2 vs. 3.7 ml x min(-1) x year(-1); range 0.16 to 16.6 vs. -3.8 to 16.0 ml x min(-1) x year(-1); P < 0.001). Significantly more patients carrying the Q allele belonged to the highest tertile of GFR decline (odds ratio = 5.7, 95% CI 4.1-7.2, P = 0.02). Levels of blood pressure, HbA1c, and albuminuria were comparable in the two genotype groups. Albuminuria (P = 0.001), mean blood pressure (P = 0.046), and PC-1 genotype (P = 0.036) independently correlated with GFR decline. Because all patients were receiving antihypertensive treatment, the faster GFR decline in the patients carrying the Q allele could be the result of reduced sensitivity to the renoprotective effect of antihypertensive therapy. PC-1 genotyping identifies type 1 diabetic patients with a faster progression of diabetic nephropathy.

Differential activation of dopamine release in the nucleus accumbens core and shell after acute or repeated amphetamine injections: a comparative study in the Roman high- and low-avoidance rat lines.

The selectively bred Roman high- and low-avoidance rats differ in emotionality and responsiveness to the motor effects of acute and repeated psychostimulant administration. These lines also show drastic differences in the neurochemical responses of their mesolimbic dopamine systems to addictive drugs. The nucleus accumbens is critically involved in the locomotor activation produced by psychostimulants and in the augmentation of this effect observed upon repeated drug administration (i.e. behavioral sensitization), although there is not a general consensus as to whether the nucleus accumbens-core or the nucleus accumbens-shell is preferentially involved in such alterations. This study was designed to evaluate the effects of acute amphetamine (0.20 mg/kg, s.c.) on dopamine output in the nucleus accumbens-shell and nucleus accumbens-core of the Roman lines under basal conditions (i.e. naïve rats) and after the repeated administration of amphetamine (1 mg/kg, s.c. x 10 days) or saline. We show that (1) in naïve rats, amphetamine caused a larger increment in dopamine output in the nucleus accumbens-shell vs the nucleus accumbens-core only in the Roman high-avoidance line; (2) repeated amphetamine elicits behavioral sensitization in Roman high-avoidance, but not Roman low-avoidance, rats; (3) in sensitized Roman high-avoidance rats, amphetamine provokes a larger increment in dopamine output in the nucleus accumbens-core, and an attenuated dopaminergic response in the nucleus accumbens-shell, as compared with Roman high-avoidance rats repeatedly treated with saline; and (4) such neurochemical changes are not observed in the mesoaccumbens dopaminergic system of the sensitization-resistant Roman low-avoidance line. We propose that (1) Roman high-avoidance and Roman low-avoidance rats differ in the vulnerability to develop psychostimulant sensitization, (2) the nucleus accumbens-core and nucleus accumbens-shell subserve distinct functional roles in this phenomenon, and (3) comparative studies in the Roman lines may provide insight into the influence of neural substrates and genetic background on the individual vulnerability to addiction.

Three-drug combination of MKC-442, lamivudine and zidovudine in vitro: potential approach towards effective chemotherapy against HIV-1.

OBJECTIVES: MKC-442 (6-benzyl-1-ethoxymethyl-5-isopropyluracil), a potent non-nucleoside reverse transcriptase inhibitor, is a promising candidate for the treatment of HIV-1 infection and is now undergoing clinical trials. We studied the in vitro activity of MKC-442 against HIV-1 replication in a three-drug combination regimen with zidovudine (ZDV) and lamivudine (3TC). METHODS: Drug-drug interactions in MT-4 cells and peripheral blood mononuclear cells (PBMC) infected with HIV-1IIIB were evaluated. The multiple drug effect analysis based on the median effect principle was applied, and the combination indices were calculated using a computer software program. The occurrence of viral breakthrough was investigated during a long-term culture of HIV-1-infected MT-4 cells. RESULTS: When MKC-442 was combined with 3TC and ZDV, they synergistically suppressed HIV-1 replication in MT-4 cells over a wide range of doses irrespective of the endpoints for synergy calculations. Similar results were also obtained in PBMC. An arbitrary combination ratio of 10:100:1 for MKC-442:3TC:ZDV showed stronger synergism than any other ratios examined. As a result of synergy in the three-drug combination, the dose of each drug could be reduced by four- to 24-fold. The three-drug combination markedly delayed or even completely suppressed HIV-1 replication at least for 40 days. Virus emerged in the presence of three drugs at lower doses, although it did not contain any amino-acid mutations in the sequenced reverse transcriptase region and did retain full sensitivity to all three drugs. CONCLUSIONS: Our results demonstrate a potential efficacy of MKC-442 in combination with 3TC and ZDV, and the three-drug combination should be considered for treatment of AIDS patients.

2-Sulfonyl-4-chloroanilino moiety: a potent pharmacophore for the anti-human immunodeficiency virus type 1 activity of pyrrolyl aryl sulfones.

The synthesis and the evaluation of cytotoxicity and anti-HIV-1 activity of new aryl pyrrolyl (8) and aryl indolyl (9) sulfones are reported. Preparation of above sulfones was achieved by reacting arylsulfonyl chlorides with substituted pyrroles and indoles or by condensing sulfonamides with 2,5-dimethoxytetrahydrofuran in glacial acetic acid according to the Clauson-Kaas method. Chemical requisites relevant to the anti-HIV-1 activity of these compounds are both a 2-sulfonyl-4-chloroanilino moiety and an alkoxycarbonyl group at position 2 of the pyrrole ring. The best activity and selectivity were obtained with ethoxycarbonyl and isopropoxycarbonyl substituents. Substitutions at the amino group of the pharmacophore moiety led to inactive products (alkylation) or weakened (acylation) anti-HIV-1 activity. Among test derivatives, 16 compounds showed EC50 values ranging between 10 and 1 microM, and five (8b',d',f',h'j') showed EC50S in the sub-micromolar range. The compounds were active against HIV-1, both wild type and AZT-resistant strains, but not against HIV-2. Moreover, in enzyme assays they potently inhibited the HIV-1 recombinant reverse transcriptase, were 10 times less active against enzymes from nevirapine- and TIBO-resistant strains, and were totally inactive against the HIV-2 recombinant enzyme. Interestingly, some compounds (8r'-y') were inactive against the recombinant reverse transcriptase while being active in tissue culture.

Synthesis and antiviral activity of 8-aza analogs of chiral [2-(phosphonomethoxy) propyl]guanines.

(R)- And (S)-8-aza-9(-)[2-(phosphonomethoxy)propyl]guanine [(R)-and (S)-8-aza-PMPG] were synthesized and tested in vitro for anti-human immunodeficiency virus (HIV) activity. The synthesis of the above compounds and of (R)-9(-)[2-(phosphonomethoxy)propyl]guanine [(R)-PMPG] was carried out through the alkylation of 8-azaguanine or guanine with (R)- and (S)-2-O(-)[(diisopropylphosphono)methyl]-1-O-(tolylsulfonyl) -1,2-propanediol followed by deprotection of the phosphonic moiety. A different, even more convenient synthesis of (R)-8-aza-PMPG starting from 2-amino-6-chloro-5-nitro-4(3H)-pyrimidinone and (R)(-)[2(-)[(diisopropylphosphono)-methoxy]propyl]amine is also reported. Both (R)-8-aza-PMPG and (R)-PMPG demonstrated anti-HIV activity in the MTT assay with EC50 values of 12 and 4.5 microM, respectively. The corresponding S enantiomers were found to be less potent. When evaluated in combination with AZT, ddI, or DABO 603, (R)-8-aza-PMPG gave additive, additive, and synergistic anti-HIV-1 effects, respectively.

Biochemical characterization of HIV-1 reverse transcriptases encoding mutations at amino acid residues 161 and 208 involved in resistance to phosphonoformate.

Mutations at amino acid residues 161 (Q161L) and 208 (H208Y) of the reverse transcriptase (RT) have been identified in HIV-1 variants which are resistant to phosphonoformate (PFA). In the present study, we report on the biochemical properties of recombinant RTs (rRTs) carrying either one or both of the above mutations. We also report on their susceptibility to PFA and to nucleoside (NRTI) and non-nucleoside (NNRTI) RT inhibitors. Like the wild-type (wt) enzyme, mutant rRTs H208Y and Q161L/H208Y showed a preference for Mg2+ over Mn2+, whereas the Q161L rRT preferred Mn2+. The three mutant rRTs showed degrees of PFA resistance which differed according to the template-primer used, and steady-state kinetic studies revealed an inverse correlation between their degree of PFA resistance, affinity for deoxynucleoside triphosphates (dNTPs) and catalytic efficiency (kcat/Km ratio). These results indicated that HIV-1 rRTs bearing mutations at codons 161 and/or 208 had altered dNTP binding sites which led to a PFA-resistant phenotype. However, unlike the corresponding mutant viruses, which are hypersensitive to 3'-azido-3'-deoxythymidine (AZT), 11-cyclopropyl-5,-11-dihydro-4-methyl-6H-dipyridol[3,2-b:2',3',-e] diazepin-6-one (Nevirapine) and (+)-(5S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)-imidazo[4,5, 1-jk][1,4]benzodiazepin-2(1H)-thione. (TIBO R82150), the mutant RTs Q161L and Q161L/H208Y were resistant to 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and as susceptible as the wt enzyme to Nevirapine and TIBO R82150. Overall, these results suggest that codons 161 and 208 of the HIV-1 RT gene are involved in substrate binding as well as in NRTI recognition, and provide more insights into the mechanism by which HIV-1 becomes resistant to PFA.