Hydrogel Containing Silibinin-Loaded Pomegranate Oil-Based Nanocapsules for Cutaneous Application: In Vitro Safety Investigation and Human Skin Biometry and Permeation Studies.

In previous studies, we developed a hydrogel formulation containing silibinin-loaded pomegranate oil nanocapsules (HG-NCSB) that had improved in vivo anti-inflammatory action in comparison to non-encapsulated silibinin. To determine skin safety and whether the nanoencapsulation influences silibinin skin permeation, NCSB skin cytotoxicity, HG-NCSB permeation in human skin, and a biometric study with healthy volunteers were conducted. The formulation of nanocapsules was prepared by the preformed polymer method while the HG-NCSB was obtained by thickening the suspension of nanocarriers with gellan gum. The cytotoxicity and phototoxicity of nanocapsules were assessed in Keratinocytes (HaCaT) and fibroblast (HFF-1) using the MTT assay. The hydrogels were characterized regarding the rheological, occlusive, and bioadhesive properties, and silibinin permeation profile in human skin. The clinical safety of HG-NCSB was determined by cutaneous biometry in healthy human volunteers. NCSB yielded better cytotoxicity results than the blank nanocapsules (NCPO). NCSB did not cause photocytotoxicity, while NCPO and the non-encapsulated substances (SB and pomegranate oil) were phototoxic. The semisolids presented non-Newtonian pseudoplastic flow, adequate bioadhesiveness, and low occlusive potential. The skin permeation demonstrated that HG-NCSB retained a higher SB amount in the outermost layers than HG-SB. In addition, HG-SB reached the receptor medium and had a superior concentration of SB in the dermis layer. In the biometry assay, there was no significant cutaneous alteration after the administration of any of the HGs. Nanoencapsulation promoted greater SB retention in the skin, averted percutaneous absorption, and made the topical use of SB and pomegranate oil safer.

Gellan gum-based hydrogel containing nanocapsules for vaginal indole-3-carbinol delivery in trichomoniasis treatment.

Trichomonas vaginalis infection is the STI most common worldwide. Indole-3-carbinol (I3C) is a phytochemical presenting promising biological activities. In this study, design, formulation, and evaluation of a vaginal hydrogel containing I3C-loaded nanocapsules for the treatment of trichomoniasis have been investigated. Nanocapsules of Eudragit® RS100 and rosehip oil containing I3C (NC-I3C) were prepared by interfacial deposition of preformed polymer method. In vitro evaluations showed that free I3C (IC50 = 3.36 µg/mL) was able to reduce the trophozoites viability at higher concentrations (3.13 and 6.25 µg/mL), while nanoencapsulation (IC50 = 2.09 µg/mL) reduced the viability at all concentrations evaluated. Comparing free and nanoencapsulated I3C, we observe that nanoencapsulation improved anti-T. vaginalis activity. In order to obtain a formulation for vaginal administration, hydrogels (HG-NC-I3C) were prepared by thickening the NC-I3C with gellan gum. HG-NC-I3C presented particle size below 195 nm, low polydispersity index (<0.2), I3C content = 0.50 ± 0.01 mg/g, pH = 7.05, non-Newtonian pseudoplastic flow behavior and exhibited mucoadhesion to cow's vaginal mucosa. Evaluation of irritation potential by chorioallantoic membrane method indicated that the formulations are considered non-irritating. Besides that, permeation through the cow's vaginal mucosa showed that nanoencapsulation promoted I3C controlled release. This way, the developed HG-NC-I3C can be considered a promising approach for trichomoniasis treatment through vaginal administration.

Discovery of a novel and selective fungicide that targets fungal cell wall to treat dermatomycoses: 1,3-bis(3,4-dichlorophenoxy)propan-2-aminium chloride.

BACKGROUND: Fungal infections are highly prevalent and are responsible for high rates of morbidity and mortality. In this context, the search for new treatment alternatives is very relevant. OBJECTIVES: Analyse chemical compounds for antifungal potential against dermatomycosis fungi. METHODS: The antifungal activity of 121 compounds, intermediates or derivatives of 1,3-bis(aryloxy)propane substituted at C-2 (111 compounds) and isothiouronium derivatives (10 compounds) was investigated through susceptibility tests, mechanism of action, toxicity and hydrogel incorporation. RESULTS: The compound 1,3-bis(3,4-dichlorophenoxy)propan-2-aminium chloride (2j) was the most active fungicide against dermatophytes and Candida spp., at very low concentrations (0.39-3.12 µg/mL), including action on resistant and multidrug-resistant clinical strains. Compound 2j has presented a promising toxicity profile, showing selectivity index >10, relative to human lymphocytes. The compound was classified as non-irritant by the HET-CAM test and did not cause histopathological alterations in pig ear skin, thus presenting an excellent perspective for topical application. 2j targets the fungal cell wall, which was confirmed by scanning electron microscopy, which also indicated the additional ability of 2j to inhibit the Candida albicans pseudohyphae formation and biofilm of Microsporum canis. Compound 2j was incorporated in a hydrogel with bioadhesive potential. The results of the human skin permeation showed that 2j remained significantly in the epidermis, ideally for the dermatomycosis treatment. CONCLUSIONS: Therefore, the compound 2j demonstrated the potential for antifungal drug development, with a action mechanism elucidated and already applied in a semisolid formulation as a new therapeutic option for fungal skin infections.

Oleic acid-containing semisolid dosage forms exhibit in vivo anti-inflammatory effect via glucocorticoid receptor in a UVB radiation-induced skin inflammation model.

The treatment of cutaneous inflammation with topical corticosteroids may cause adverse effects reinforcing the need for therapeutic alternatives to treat inflammatory skin disorders. We investigated the anti-inflammatory effect of oleic acid (OA), a fatty acid of the omega-9 (ω-9) family, and we point out it as an alternative to treat inflammatory skin disorders. OA was incorporated into Lanette®- or Pemulen® TR2-based semisolid preparations and the pH, spreadability, rheological behavior and in vivo anti-inflammatory performance in a UVB radiation-induced skin inflammation model in mice were assessed. The anti-inflammatory activity was verified after single or repeated treatment of the mouse ear following the UVB. The OA action on glucocorticoid receptors was investigated. Both semisolids presented pH values compatible with the deeper skin layers, appropriate spreadability factors, and non-Newtonian pseudoplastic rheological behavior. Pemulen® 3% OA inhibited ear edema with superior efficacy than Lanette® 3% OA and dexamethasone after a single treatment. Pemulen® 3% OA and dexamethasone also reduced inflammatory cell infiltration. After repeated treatments, all formulations decreased the ear edema at 24 h, 48 h and 72 h after UVB. OA in semisolids, especially Pemulen® TR2-based ones, presented suitable characteristics for cutaneous administration and its anti-inflammatory activity seems to occur via glucocorticoid receptors. OA was also capable to reduce croton oil-induced skin inflammation. Besides, the ex vivo skin permeation study indicated that OA reaches the receptor medium, which correlates with a systemic absorption in vivo. The natural compound OA could represent a promising alternative to those available to treat inflammatory skin disorders.

Nanocapsules improve indole-3-carbinol photostability and prolong its antinociceptive action in acute pain animal models.

This study aimed the development of nanocapsules (NCs) for oral indole-3-carbinol (I3C) administration and evaluation of antinociceptive potential of this compound in its two forms, free and nanoencapsulated, using acute pain models. NCs showed adequate physicochemical characteristics and protected the I3C against UVC radiation exposure. It was observed no chemical bond between I3C and polymer by FTIR. Besides, X-ray and DSC analysis suggested that I3C was molecularly dispersed in NCs. The dialysis bag technique showed that almost 100% of the compound was released from NCs at 360min. Mathematical modeling demonstrated that this release occurred in two rates, with an initial burst effect followed by a slower release of I3C. Regarding the in vivo analysis, time-response curve showed that both forms of I3C caused an inhibition in inflammatory phase of nociception induced by formalin and increased the latency response in hot plate test. Interestingly, NCs were able to prolong the I3C effect in both tests. Furthermore, in dose-response curve, only I3C in its nanoencapsulated form presented effect on inflammatory phase of the formalin test. In conclusion, NCs to I3C incorporation presented adequate nanometric characteristics and prolonged its antinociceptive action in acute pain models tested.

Enhanced photostability, radical scavenging and antitumor activity of indole-3-carbinol-loaded rose hip oil nanocapsules.

This study aimed to develop poly(ε-caprolactone) nanocapsules loaded with indole-3-cabinol (I3C) using rose hip oil (RHO) or medium chain triglycerides (MCT) as oil core. In vitro radical scavenging activity (DPPH method), hemolysis, and antitumor effects on breast (MCF-7) and glioma (C6) cells were conducted. Preformulation evaluations revealed that RHO is suitable to prepare the nanocapsules considering the log P determination and dissolution/swelling experiments of polymer films. The nanocapsules were prepared and presented adequate physicochemical characteristics as mean size around 250nm, polydispersity index values <0.2, zeta potential negative values and I3C encapsulation efficiency around 42%, without any influence of the oil core (RHO or MCT) on these parameters. However, the photodegradation study demonstrated that RHO nanocapsules showed less degree of I3C degradation in comparison to MCT nanocapsules. The in vitro release profile showed that both nanocapsule suspensions demonstrated an initial burst effect followed by a prolonged I3C release. In addition, the formulations were considered hemocompatibles at 10µg/mL and showed an enhanced radical scavenging activity in comparison to free I3C. Moreover, nanocapsules prepared with RHO increased about two times the antitumor effect of I3C on MCF-7 and C6 cells without significant reduction of astrocyte cell viability. In conclusion, nanocapsule formulations developed in this study might be considered promising for cancer treatment.

Detection of GAD65 autoreactive T-cells by HLA class I tetramers in type 1 diabetic patients.

Type 1 diabetes (T1D) is an autoimmune disease, in which pancreatic beta cells are destroyed in genetically predisposed individuals. While the direct contribution of autoantibodies to the disease pathogenesis is controversial, it is generally recognised that the mechanism of beta cell destruction is mediated by autoreactive T cells that had escaped the thymic selection. We aimed to design a method to detect circulating CD8+ T cells autoreactive against an epitope of the glutamic acid decarboxylase autoantigen, isoform 65 (GAD65) ex vivo in T1D patients by using HLA class I tetramers. Low frequencies of GAD65 peptide-specific CD8+ cytotoxic T lymphocytes were detected in peripheral blood lymphocytes (PBMC) of normal controls after GAD65 peptide-specific stimulation. Conversely, their frequencies were significantly higher than in controls in PBMC of T1D patients after GAD65 peptide stimulation. These preliminary data are encouraging in order to develop a reliable assay to be employed in large-scale screening studies.

Proteomic investigation in A549 lung cell line stably infected by HPV16E6/E7 oncogenes.

BACKGROUND: Data have accumulated implicating the involvement of oncogenic human papillomaviruses (HPVs) in bronchial carcinogenesis. We recently described the presence of oncogenic HPV transcripts in non-small cell lung cancers. OBJECTIVE: To investigate the role of oncogenic HPVs in lung carcinogenesis. MATERIAL AND METHODS: The lung cell line A549 stably infected with HPV16E6, HPV16E7 and HPVE6/E7 constructs was used to investigate the protein profile changes associated with the expression of these oncogenes. Replicated two-dimensional gel electrophoresis gels from uninfected and stably HPV16E6-, E7-, and E6/E7-infected A549 cells were compared for changes in protein profile. Protein identification was achieved by peptide mass fingerprinting by MALDI-TOF-MS and nLC-ESI-Q-TOF-MS/MS peptide ladder sequencing. RESULTS: We identified 17 different polypeptides whose average normalized spot intensity was statistically significant (p < 0.05) and differed by 2-fold. Relationships between differentially expressed proteins and the HPV-induced infection mechanism have been clustered by knowledge-base database functional association network analysis. CONCLUSION: The impact of Hsp27, annexin III, annexin IV, Gp96 and TPT1 on the cellular response mechanism to HPV infection is presented and discussed.

Identification of an adult stem/progenitor cell-like population in the human thyroid.

There is evidence that tissue-specific stem cells reside in certain adult tissues. Their specific properties remain elusive, because they are rare and heterogeneous in parent tissues; furthermore, technical difficulties have been encountered in the identification and characterization of their progeny. The aim of this study was to isolate stem/progenitor cells from the human thyroid. We devised a method based on the enzymatic digestion of fresh surgical thyroid specimens, followed by culture of cells in the presence of epidermal growth factor and basic fibroblast growth factor. We also used markers that identify and characterize these cells. Spheroids with self-replicative potential were obtained from all thyroid specimens. The isolated population contained a subset of CD34+ CD45- cells and it was able, in differentiation conditions, to generate follicles with thyroid hormonal production. In support of the plasticity concept, we obtained evidence that, when most freshly isolated spheroids were co-cultured with a neuroblastoma cell line, they produced progeny expressing the neuronal marker beta-tubulin III. Spheroids were also able to undergo adipogenic differentiation when cultured in adipogenic medium. We conclude that a predominant functional type of stem/progenitor cell exists within the thyroid, with an intrinsic ability to generate thyroidal cells and the potential to produce non-thyroidal cells.

Detection of oncogenic DNA viruses in colorectal cancer.

BACKGROUND: As a part of our search for oncogenic viruses as potential etiological agents in human malignancies, our studies on human papillomaviruses (HPV) were extended to analysis of the 3 polyomaviruses (SV40, BKV and JCV) in colorectal carcinomas. PATIENTS AND METHODS: Archival tumour samples from 71 patients with colorectal cancer were analyzed for the sequences of SV40, BKV, JCV and HPV using PCR-based techniques. HPV genotypes were determined using sequencing and reverse blot hybridization (InnoLipa). RESULTS: Amplification of BKV and JCV with the primer pair PEP-1 and PEP-2 and subsequent restriction digestion of the amplified products with BamH I disclosed BKV in 6/66 (9%) of the samples, whereas none contained JCV. SV40 was amplified in 10/66 (15.1%) samples and confirmed by sequencing analysis. In pair-wise analysis for co-infections, the samples were significantly different in their BKV-JCV and JCV-SV40 status, in contrast to their BKV-SV40 co-infection status. HPV DNA was detected in 22/66 (33.3%) of the samples analysed with either the MY09/11 or SPF primer mix. Of these 22 HPV infections, 7 were single-type infections and 15 contained multiple HPV types. HPV detection or type distribution showed no relationship to the gender of the patients or histological grade of the tumour. HPV status was not significantly related to detection of BKV, JCV or SV40. Similarly, in pair-wise analysis for co-infections, the samples were significantly different in their status of HPV-BKV (p=0.0006), HPV-JCV (p=0.0001), and HPV-SV40 (p=0.019), implicating that HPV and the 3 polyomaviruses are rarely detected concomitantly in the same samples. CONCLUSION: Taking the known molecular mechanisms of action of these individual viruses, there is a chance that these viruses could alter the mechanisms of cell cycle control and inhibit apoptosis, thus potentially causing chromosomal instability and promoting colorectal oncogenesis.

Human papillomavirus infections in lung cancer. Detection of E6 and E7 transcripts and review of the literature.

Lung cancer is the leading cause of cancer related death in Western countries. Several factors have been implicated in its aetiology: cigarette smoking, environmental pollution, asbestos and genetic factors. The possible involvement of human papillomavirus (HPV) in bronchial squamous cell lesions was first suggested in 1979 by Syrjänen. Since then, several studies have confirmed the presence of HPV DNA in about 20% of lung cancer cases examined, with HPV16 and 18 as the two most frequently found oncogenic viral types. More recently, these data have been supported by the detection of E6 and E7 transcripts in HPV-positive lung cancer cases, reinforcing the hypothesis that oncogenic HPVs could act as cofactors in bronchial carcinogenesis. This published literature is briefly reviewed and new data of the authors on detection of E6 and E7 transcripts in lung cancer samples are presented.

Detection of oncogenic viruses SV40, BKV, JCV, HCMV, HPV and p53 codon 72 polymorphism in lung carcinoma.

As a part of our continuous search for oncogenic viruses in bronchial cancer, we extended our HPV studies to analyse also SV40, BKV, JCV and HCMV sequences in bronchial cancer and related these data with p53 codon 72 polymorphism. Fresh tumor samples from 78 patients with lung cancer were analysed for SV40, BKV, JCV, HCMV and HPV sequences by PCR. HPV genotypes were determined using reverse blot hybridization and sequencing, and all HPV-positive tumors were tested for the presence of E6/E7 transcripts by RT-PCR. All samples were analysed for p53 codon 72 polymorphism, using PCR-based RFLP method. Of the 78 cases studied, 11 (14.1%) were positive for T-Ag gene of SV40, while BKV and JCV sequences were both amplified in 1 tumor only. Altogether, 10/78 lesions were HPV-positive; six HPV16, one HPV31, two HPV6/53 and one HPV16/18. All HPV DNA-positive samples except one also expressed E6 and E7 transcripts. HCMV was amplified in 18 (23%) cases. RFLP analysis of p53 codon 72 revealed 32 homozygotes for arg/arg allele (50.8%), 26 heterozygotes for arg/pro allele (41.3%), and 5 homozygotes for pro/pro allele (7.9%). P53 codon 72 polymorphism was not significantly different between cases (n=63) and controls (n=50) (p=0.455), among virus positive and negative patients, nor was it related to HPV genotypes (p=0.384), expression of E6 (p=0.384) and E7 oncogenes (p=0.293). Of all possible combinations of virus co-detection, only SV40-HCMV association was statistically significant (OR=5.500, 95%CI 1.43-21.02; p=0.015). Taken the known mechanisms of these individual viruses, there is a chance that these viruses could affect cell cycle control and inhibit apoptosis, thus potentially causing genetic instability and promote oncogenesis.

The p53 codon 72 arg/arg homozygous women in central Italy are at increased risk for HPV infections.

BACKGROUND: The oncoprotein E6 binds to and degrades the p53 tumor suppressor protein, with different efficacy depending on the p53 codon 72 (arg/pro) polymorphism. The arg/arg allele has been shown to increase the risk for cervical cancer. MATERIALS AND METHODS: Fifty-eight women infected with HPV and 32 normal controls were analyzed by restriction fragment length polymorphism to detect arg/arg or arg/pro alleles. RESULTS: The allele frequencies in HPV-positive women were: arg/arg 47/58 (81%); arg/pro 9/58 (15.5%) and pro/pro 2/58 (3.4%), while those in controls were: arg/arg 19/32 (59%); arg/pro 10/32 (31.2%) and pro/pro 3/32 (9.3%) (Fisher's exact test, p = 0.068). The risk of having HSIL in arg/arg homozygous patients had odds ratio (OR) = 1.33 (95% CI 1.12-1.58, p = 0.628). Women with the arg/arg phenotype were at significantly increased risk for HPV infection; OR = 2.93 (95% CI 1.11-7.66, p = 0.028). Being homozygous arg/arg also substantially increased the risk of HR-HPV infection, with OR = 3.84 (95% CI 0.71-20.57, p = 0.128), whereas heterozygosity for arg/pro was protective against HR-HPV; OR = 0.186 (95% CI 0.03-1.04, p = 0.074). Allele frequencies in women with different HPV types were not significantly different, however (p = 0.174). CONCLUSION: These data suggest that arg/arg homozygous patients are at increased risk for HR-HPV infections.

Comparison of DNA sequencing and Roche Linear array in human papillomavirus (HPV) genotyping.

BACKGROUND: Human papillomavirus (HPV) is the etiological agent of cervical cancer. HPV genotyping is important to determine the presence of high-risk types. Recently, a new HPV genotyping method, the Roche Linear array genotyping test, was introduced and is compared here with a sequencing-based HPV genotyping system. MATERIALS AND METHODS: A series of 102 women (age range 30-55 years) shown to be HPV DNA-positive by PCR were typed by sequencing and the Linear array genotyping assay. RESULTS: The sequence analysis revealed the presence of 80 single high-risk types and 22 single low-risk types. With the Linear array, single infections were found in 46 cases, double infections in 37 cases, triple infections in 12 cases, and more than three in 6 cases. One case positive by sequencing gave a negative result by Linear array. Altogether, a concordant single genotype was found in 93 (91.2%) out of the 102 cases and the single-type concordance between the two assays was significant (Spearman rho = 0.849, p = 0.0001; intraclass correlation coefficient (ICC) (ICC = 0.924, 95% CI 0.888-0.949) (p = 0.0001). The majority of the disparate results were due to the detection of multiple types by the Linear array. CONCLUSION: The Roche Linear array is a highly accurate assay for HPV genotyping. This is particularly true in the presence of multiple infections which DNA sequencing is unable to resolve.

Detection and expression of human papillomavirus oncogenes in non-small cell lung cancer.

Human papillomavirus (HPV) has been found in lung cancer cases with variable frequency. In the present study, we analysed a series of 38 patients with non-small cell lung cancer (NSCLC) (21 paraffin-embedded archival samples and 17 fresh surgical specimens) for the presence of E6 and E7 oncogenes of HPV16, 18 and 31. Eight of the tumours were positive (21%): six HPV16, one HPV16+18, and one HPV31. The normal tissue surrounding the HPV-positive tumour was negative for the presence of the virus. Sequencing analysis of URR, of HPV16, which was the most frequently found HPV type in our cases, showed an adenosine deletion at nucleotide 7861 (E2-binding site) in four out of six patients. Sequencing of the entire E6 and E7 genes of HPV16 showed a T to G transition at nucleotide position 350 of E6, in all examined cases. This mutation is associated to the European variant of HPV16. Analysis of E6 and E7 transcripts was performed on the six fresh surgical specimens infected by HPV16. Our study showed that all of the tumours investigated, except one, contained E6 and E7 transcripts. Only in one case could we identify an unspliced form of the E6 transcript. Our results strengthen the relationship between HPV and NSCLC and support the hypothesis that HPV infection could play a role in bronchial carcinogenesis.

UDP-glucuronosyltransferases 1A expression in human urinary bladder and colon cancer by immunohistochemistry.

UDP-glucuronosyltrasferases (UGTs) are detoxifying enzymes, which convert endogenous substrates, dietary constituents and potential carcinogens to inactive hydrophilic glucuronides. Although the liver is considered the most important organ for glucuronidation, UGTs are also expressed in extrahepatic tissues. Since UGTs may be important to protect cells from cancer in organs naturally exposed to potential carcinogens such as smoking and diet derivatives, we investigated the UGT expression in normal and malignant tissues from urinary bladder and large intestine. The study was carried out by immunohistochemistry, using an antiserum recognizing all the UGT1A isoforms. Our results showed that UGTs were highly expressed on the surfaces of the normal bladder as well as in normal large bowel mucosa. The neoplastic counterpart showed a general protein down-regulation associated with a different cellular localization. We found that 3/11 papillary and 3/6 invasive urothelial carcinomas were virtually negative, whereas 2/2 papillomas and 8/11 papillary bladder carcinomas still expressed the protein. In colon cancer the enzyme down-regulation was even more dramatic. In fact, a faint diffused cytoplasmic expression or scattered cell positivity was observed only in adenomas with low grade dysplasia (5/5) and in 2/11 carcinomas. Interestingly, 5/5 adenomas with high grade dysplasia, 9 carcinomas, the lymph nodes and liver metastases were UGT-negative, suggesting that the loss of UGT is associated with the early phase of neoplastic transformation. Based on our results we suggest that UGTs constitutive expression in the normal mucosa could protect these organs from carcinogens released in the bladder or introduced directly with the diet in the colon.

Equivalent immunogenicity of the highly attenuated poxvirus-based ALVAC-SIV and NYVAC-SIV vaccine candidates in SIVmac251-infected macaques.

Therapeutic immunization of HIV-1-infected individuals may induce and/or enhance HIV-1-specific immune responses and decrease the dependency on antiretroviral drug treatment. However, repeated immunizations with live-recombinant vectors may induce vector-specific immune responses that interfere with the elicitation of vigorous immune responses to the desired antigen. Therefore, the use of mixed-modality vaccinations may be necessary to induce sustained virus-specific immune responses in HIV-1-infected individuals treated with antiretroviral therapy (ART). Thus, the relative immunogenicity of various vaccine modalities needs to be assessed. Here we compared the immunogenicity of two vaccine candidates, the canarypox-based ALVAC-SIV-gag-pol-env (ALVAC-SIV-gpe) and the vaccinia-based NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe), in rhesus macaques infected with SIVmac251 and treated with ART by 2 weeks postinfection. Both ALVAC-SIV-gpe and NYVAC-SIV-gpe vaccine candidates induced and/or enhanced a virus-specific CD8+ T cell response to a similar extent, as demonstrated by tetramer staining of Gag-specific CD8+ T cells. Similarly, both vaccines elicited comparable lymphoproliferative responses (LPRs) to the SIV p27 Gag and gp120 Env proteins. Thus, both these vaccine modalities alone or in combination may be suitable candidate vaccines for immune therapy of HIV-1-infected individuals.