Targeted delivery of anti-cancer growth inhibitory peptides derived from human alpha-fetoprotein: review of an International Multi-Center Collaborative Study.

The alpha-fetoprotein derived growth inhibitory peptide (GIP) is a 34-amino acid peptide composed of three biologically active subfragments. GIP-34 and its three constituent segments have been synthesized, purified, and studied for biological activity. The GIP-34 and GIP-8 have been characterized as anticancer therapeutic peptides. An multicenter study was initiated to elucidate the means by which these peptide drugs could be targeted to tumor cells. The study first established which cancer types were specifically targeted by the GIP peptides in both in vitro and in vivo investigations. It was next demonstrated that radiolabeled peptide ((125)I GIP-34) is specifically localized to rodent breast tumors at 24 h post-injection. The radionuclide studies also provided evidence for a proposed cell surface receptor; this was confirmed in a further study using fluorescent-labeled GIP-nanobeads which localized at the plasma membrane of MCF-7 breast cancer cells. Finally, it was readily demonstrated that GIP conjugated to either fluorescein or doxorubicin (DOX) underwent tumor cell uptake; subsequently, DOX-GIP conjugates induced cytotoxic cell destruction indicating the utility of GIP segments as cancer therapeutic agents. Following a discussion of the preceding results, a candidate cell surface receptor family was proposed which correlated with previous published reports for a putative AFP/GIP receptor.

Hantavirus infection in people inhabiting a highly endemic region of the Gran Chaco territory, Paraguay: association with Trypanosoma cruzi infection, epidemiological features and haematological characteristics.

The seroprevalences of anti-hantavirus antibodies were determined in 712 individuals (551 Indians, 140 Mennonites of German ancestry, and 21 Paraguayans of Spanish ancestry) inhabiting a region of western Paraguay in the Gran Chaco territory of South America. The overall seroprevalence of hantavirus infection among the 712 subjects, who were aged 2-80 years, was 42.7% (45.2% in the Indians and 34.2% in the non-Indians). Of the 672 subjects also checked for antibodies against Trypanosoma cruzi, 226 (33.6%) were seropositive for this protozoan parasite. The results of a multivariate regression analysis indicated that, after adjusting for age, sex, setting of residence (rural/urban) and infection with the human T-cell leukaemia/lymphoma virus type II (HTLV-II), a T. cruzi-seropositive individual was 1.73 times more likely to be hantavirus seropositive than a T. cruzi-seronegative individual. Living in a rural setting increased the risk of being hantavirus seropositive 2.17-fold. In both the Indians and non-Indian subpopulations, hantavirus seroprevalence increased with age in both sexes, but only in the non-Indian supopulation was this increase significantly greater in males than in females. Hantavirus seropositivity was significantly associated with thrombocytosis, even after adjusting for the relevant confounders.

Classic Kaposi's sarcoma associated with human herpesvirus 8 infection in a 13-year-old male: a case report.

PURPOSE: Classic Kaposi's sarcoma (KS) is rare in children. Although its etiology is not fully understood, human herpesvirus 8 (HHV-8) is present in the angiogenic lesions. We report an HIV-negative, 13-year-old patient of Sicilian descent with HHV-8-associated classic KS to facilitate the diagnosis and treatment of this entity in children. EXPERIMENTAL DESIGN: DNA was extracted from the skin specimen of the patient and analyzed via PCR assay and Southern blot hybridization for HHV-8 DNA. The amplified HHV-8 DNA was cloned, sequenced, and compared with the prototype HHV-8-KS330/BAM. RESULTS: The patient presented with purpuric lesions on the distal lower extremities and the tip of his nose, associated with thrombocytopenia and leukopenia, suggesting an immune-mediated cytopenia. While on prednisone, he developed marked vascular proliferation in the groins. Biopsy of the skin lesions showed KS, and HHV-8 was detected in the tissues by PCR. Sequence analysis of the amplified DNA was homologous to the prototype HHV-8-KS330/BAM. His HHV-8 strain was the A subgroup, the type associated with Mediterranean classic KS. Stopping prednisone and treatment with IFN-alpha and IgG resulted in regression of the groin lesions. CONCLUSIONS: This report emphasizes the importance of recognizing classic KS in children and avoiding immunosuppressive therapies in indolent classic KS. The diagnostic and therapeutic strategies were effective and well tolerated.

Prevalence of HTLV-I-associated T-cell lymphoma.

In order to assess the prevalence rate of HTLV-1-associated T-cell lymphomas and human retrovirus infection in general, approximately 21,000 individuals representing various patient populations, retroviral risk groups, and blood donors were examined for HTLV-I, HTLV-II, HIV-1, or HIV-2 infection using serologic and PCR assays. The prevalence rates among volunteer blood donors were 0.02% and 0% for HTLV and HIV, respectively. Significantly increased HTLV prevalence rates were observed among paid blood donors, African American health care clinic patients, Amerindians, recipients of HTLV-positive cellular blood products, intravenous drug users, sexual contacts and family members of HTLV-positive people, and patients with primary thrombocytosis and other-than-low-grade non-Hodgkin's lymphoma (NHL). Among some of these groups there were significant differences in the prevalence of HTLV-I versus HTLV-II. The eight HTLV-positive NHL patients all had mature, high-grade, CD4+ T-cell lymphomas with clonally integrated HTLV-I, for a prevalence of 4% among other-than-low-grade NHL patients. Seven of the eight died from their disease within 2 years despite treatment. Interestingly, two groups at risk for HTLV infection, namely needle stick victims and recipients of HTLV-infected and/or pooled plasma products, showed no evidence for infection. Significantly increased HIV-1 prevalence was observed among paid blood donors, African Americans, homosexuals, female prostitutes, hemophiliacs, and other-than-low-grade NHL patients. Only one patient was infected with HIV-2. Of the nine HIV-positive, other-than-low-grade NHL patients, seven HIV-1 positives had B-cell lymphomas, one HIV-1 positive had an HTLV-I-positive CD4+ T-cell lymphoma, and one infected with HIV-2 had a CD4+ T-cell lymphoma that was HTLV negative. The data indicate that HTLV-I lymphoma, while uncommon, is not necessarily rare among other-than-low-grade NHL cases in the United States and, given its poor prognosis, should probably be studied separately in clinical trials.

The complete genomic sequence of a BLV strain from a Holstein cow from Argentina.

DNA was extracted from the peripheral blood of a seropositive, PCR-positive, BLV-infected Holstein cow (No. 38) from Argentina. The DNA was amplified via PCR with a series of overlapping primers encompassing the entire BLV proviral DNA. The amplified BLV ARG 38 DNA was cloned, sequenced, and compared phylogenetically to three other full-length BLV sequences. Characterization of its deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.

Comparative performances of an HTLV-I/II EIA and other serologic and PCR assays on samples from persons at risk for HTLV-II infection.

BACKGROUND: HTLV-I and HTLV-II are related exogenous pathogenic human retroviruses. Until recently, ELISAs based on HTLV-I antigens have been used to screen for the presence of HTLV-I or -II antibodies. The HTLV-I-based assays have not been as sensitive in detecting antibodies to HTLV-II as in detecting antibodies to HTLV-I. The Abbott HTLV-I/HTLV-II ELISA uses a combination of HTLV-I and HTLV-II antigens to detect antibodies to the whole HTLV group. The performance of this ELISA was compared to that of several HTLV-I-based serologic assays and an HTLV-II PCR assay in cohorts of South American Indians and New York City IV drug users (IVDUs) in whom HTLV-II is endemic. STUDY DESIGN AND METHODS: Sera from 429 South American Indians and New York City IVDUs were evaluated for HTLV antibodies by the use of three ELISAs (rgp21-enhanced HTLV-I/II, Cambridge Biotech; Vironostika HTLV-I/II, Organon Teknika; and HTLV-I/HTLV-II, Abbott), and a Western blot (WB) assay. Peripheral blood leukocyte DNA from each person was analyzed for HTLV-I and HTLV-II pol DNA via PCR. The HTLV-II PCR-positive samples were further subtyped via cloning and sequencing and/or oligomer restriction. RESULTS: Two hundred four samples (48%) were positive for HTLV-II by serologic and/or PCR assays. All of the positive samples from the Indians and approximately one-third of the positive samples from the IVDUs were of the HTLV-IIB subtype. Comparative analyses indicate that the sensitivity and specificity of the various assays were: PCR, 98 and 100 percent; Abbott HTLV-I/HTLV-II, 78 and 95 percent; Cambridge Biotech HTLV-I/II, 76 and 96 percent; Vironostika HTLV-I/II, 71 and 98 percent; and WB, 73 and 100 percent, respectively. CONCLUSION: There were no significant differences among the sensitivities and specificities of the HTLV-I/II ELISAs (p values, 0.056-0.438). The WB and PCR assays were much more specific than the other serologic assays (p

Lack of BLV and PTLV DNA sequences in the majority of patients with large granular lymphocyte leukaemia.

The primate T-cell lymphoma/leukaemia viruses (PTLV) and bovine leukaemia virus (BLV) comprise a unique genus of retroviruses, infection with which induces seroreactivity in the host against conserved epitopes in their p24 gag and gp21 env cognate proteins. Herein, we have confirmed this serocrossreactivity. Patients with large granular lymphocyte (LGL) leukaemia have frequent seroreactivity to the p24 and gp21 env proteins of human T-cell lymphoma/leukaemia virus I (HTLV-I), one of the species in the genus. However, only a small minority of patients are actually infected with prototypic HTLV-I or HTLV-II, another species within the group. In an attempt to determine whether LGL leukaemia might be associated with other members of the PTLV/BLV genus, we examined the peripheral blood mononuclear cell DNA of 22 HTLV p24 and/or gp21 seropositive LGL leukaemia patients via PCR using degenerate and specific primer pair/probe systems capable of detecting all known members of the PTLV/BLV genus. None of the samples was positive. These data indicate that although HTLV-II may be associated with some cases of LGL leukaemia most patients are not infected with a PTLV or BLV virus.

Cytokine replacement in patients with HIV-1 non-Hodgkin's lymphoma: the rationale for low-dose interleukin-2 therapy.

PURPOSE: The drastic increase in the incidence of non-Hodgkin's lymphoma in patients infected with HIV-1 is testimony to the fact that our immune system is critical for the prevention of certain malignancies. Preclinical and clinical studies were conducted to (1) gain further insight into defects in immunity that can lead to malignant transformation and (2) determine if certain immune deficiencies could be corrected by cytokines delivered at doses that result in near-physiologic concentrations in vivo. METHODS: We have used the severe combined immune deficient mouse engrafted with human peripheral blood leukocytes from healthy individuals who are seropositive for the Epstein-Barr virus to study the spontaneous development of malignant Epstein-Barr virus-positive human B-cell lymphoproliferative disorder. RESULTS: We have demonstrated in this model that, in the absence of CD4+ T cells, cytokine replacement with low-dose interleukin (IL)-2 therapy can prevent Epstein-Barr virus-positive human B-cell lymphoproliferative disorder by interacting with mouse natural killer and human CD8+ T cells. We review our clinical experience with administration of low-dose IL-2 therapy in patients with HIV-1-related cancer, noting minimal toxicity and significant immune modulation. We provide evidence that this therapy can favorably alter the type 1 cytokine profile in vivo in these patients, and improve the cellular response to infectious insults in vitro. CONCLUSION: Early clinical studies with low-dose IL-2 therapy in patients with HIV-1-related lymphoma suggest that this therapy may have a role in the prevention and treatment of this disease.

Multiplex detection of four pathogenic retroviruses using molecular beacons.

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100, 000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.

Lymphoma presenting as a soft tissue mass. A soft tissue sarcoma simulator.

Lymphoma presenting as a soft tissue mass is rare and thus may be confused with the more common soft tissue sarcoma. No previous analysis of the clinical and radiologic features of lymphomas presenting as soft tissue masses is available because most of the cases reviewed are from the pathology literature. Four patients with diagnoses of extranodal lymphomas of the soft tissues were reviewed retrospectively with respect to their clinical features, primary tumor characteristics, stage, radiographic characteristics, treatment, and followup. Mean age was 72.5 years (range, 52-85 years). The soft tissue mass occurred in the thigh (three cases) and shoulder (one case). The median size of the soft tissue mass was 6.7 cm (range, 2-15 cm) in the largest dimension, as measured on magnetic resonance imaging. These patients each had evidence of lymphadenopathy at the time of diagnosis. Lactate dehydrogenase was increased significantly in two cases and increased slightly in two other cases. One case was Stage II(E) at presentation, one was Stage III(E), and two were Stage IV. All were B cell immunophenotype. All patients died between 2 and 24 months after diagnosis, despite the use of Cytoxan, vincristine, adriamycin, and prednisone chemotherapy in each case. Clinical and radiographic features that favor extranodal soft tissue lymphoma over sarcoma include pain and tenderness, lymphadenopathy (particularly when confluent radiologically), ipsilateral extremity swelling, and elevated lactate dehydrogenase.

Prognostic significance of K-ras codon 12 mutations in patients with resected stage I and II non-small-cell lung cancer.

PURPOSE: The aim of this study was to investigate the prognostic importance of codon 12 K-ras mutations in patients with early-stage non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: We identified 260 patients with surgically resected stage I (n = 193) and stage II (n = 67) NSCLC with at least a 5-year follow-up. We performed polymerase chain reaction analysis of DNA obtained from paraffin-embedded NSCLC tissue, using mutation-specific probes for codon 12 K-ras. RESULTS: K-ras mutations were detected in 35 of 213 assessable specimens (16.4%). K-ras mutations were detected in 27 of 93 adenocarcinomas (29.0%), one of 61 squamous cell carcinomas (1.6%), five of 39 large-cell carcinomas (12.8%), and two of 20 adenosquamous carcinomas (10%) (P = .001). G to T transversions accounted for 71% of the mutations. There was no statistically significant difference in overall survival for all patients with K-ras mutations (median survival, 39 months) compared with patients without K-ras mutations (median survival, 53 months; P = .33). There was no statistically significant difference in overall or disease-free survival for subgroups with stage I disease, adenocarcinoma, or non-squamous cell carcinoma or for specific amino acid substitutions. The median survival time for stage II patients with K-ras mutations was 13 months, compared with 38 months for patients without K-ras mutations (P = .03). CONCLUSION: Codon 12 K-ras mutations were more common in adenocarcinomas than in squamous cell carcinomas. For the subgroup with stage II NSCLC, there was a statistically significant adverse effect on survival for the presence of K-ras mutations. However, when the entire group was considered, the presence of K-ras mutations was not of prognostic significance in this cohort of patients with resected early-stage NSCLC.

Comparison of human immunodeficiency virus 1 DNA polymerase chain reaction and qualitative and quantitative RNA polymerase chain reaction in human immunodeficiency virus 1-exposed infants.

BACKGROUND: HIV-1 RNA PCR is a widely available and sensitive assay but has not been studied for use in early diagnosis of HIV-1 infection in infants. METHODS: Research HIV-1 DNA PCR and HIV-1 RNA PCR were performed on peripheral blood mononuclear cells and plasma, respectively, from 284 blood samples from 204 infants. A commercially available HIV-1 quantitative RNA PCR was also performed on plasma from the 132 samples from HIV-1-infected infants and 22 of the samples from HIV-1-uninfected infants. RESULTS: Sensitivities of all assays varied with infant age. HIV-1 DNA PCR had a sensitivity of 27% in the < or = 3-week age group (n = 11) whereas qualitative and quantitative RNA PCR had sensitivities of 64 and 55%, respectively (P not significant). Each assay had a sensitivity of 96.2% at 4 to 6 weeks (n = 26) and 100% at > or = 7 weeks of age (n = 95). Specificity of HIV-1 DNA PCR for all age groups was 100%, whereas specificities of qualitative and quantitative RNA PCR assay were 96.1 and 95.5%, respectively. CONCLUSIONS: HIV-1 RNA PCR may offer a slight advantage in sensitivity over DNA PCR in the diagnosis of HIV infection in young infants. Positive RNA results can be found in a small number of infants who are not HIV-1-infected. HIV-1 RNA detection should not be routinely used alone for the diagnosis of HIV infection in young infants.

The complete genomic sequence of an HTLV-II isolate from a Guahibo Indian from Venezuela.

A polyclonal CD3(+), CD8(+) T-cell line, G2, was derived from the peripheral blood of a seropositive, PCR-positive, HTLV-IIB infected Guahibo Indian from Venezuela. The cell line is productively infected with HTLV-IIB. The entire HTLV-II G2 proviral DNA was sequenced via PCR using overlapping HTLV-II primer pairs. Phylogenetic analyses indicate that HTLV-II G2 is the most divergent HTLV-IIB strain identified to date. Characterization of its deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.

HTLV-II risk factors in Native Americans in Florida.

Earlier virologic studies established that human T-cell lymphotropic virus type II (HTLV-II) is the predominant retrovirus type found among Seminole Indians in southern Florida. We studied 46 members of the Seminole tribe living on 3 reservations to determine the risk factors for HTLV-II and to investigate disease association with the virus. The donors' plasma samples were evaluated with the enzyme-linked immunosorbent and Western blot assays. DNA extracted from their peripheral blood mononuclear cells were analyzed by type-specific polymerase chain reaction amplification and detection of the HTLV pol gene using the primer pair SK110/SK111, and the probes SK112 or SK188. One of 46 (2%) subjects was identified as HTLV-I positive and 11 (24%) were identified as HTLV-II positive. Restriction fragment length polymorphism and sequence analyses indicated that all of the HTLV-II strains were subtype b. Mitochondrial DNA analyses indicated that all of the HTLV-II-positive subjects had an Amerindian haplotype. HTLV-II was more prevalent in Indians who were >45 years of age or female, had multiple sex partners or had received a blood transfusion. However, only the latter risk factor was statistically significant. Three of the HTLV-II-positive Indians demonstrated signs and symptoms of an ataxic neuropathy. The data support that HTLV-IIb is endemic among the Seminoles and that they will be a key population for further virologic studies.

Detection of human herpesvirus 8 DNA sequences in tissues and bodily fluids.

Human herpesvirus 8 (HHV-8) has been proposed as a sexually transmitted etiologic agent of Kaposi's sarcoma (KS). In this study, by use of a sensitive polymerase chain reaction assay, HHV-8 DNA was detected in the skin lesions (92%), normal skin (23%), peripheral blood mononuclear cells (PBMC) (46%), plasma (7%), saliva (37%), and semen (12%) but not stool samples from KS patients. The average number of HHV-8 copies per microgram of positive target DNA was 64, 000, 9000, 40, 33,000, and 300 for skin, PBMC, plasma, saliva, and semen samples, respectively. Only 1 non-KS donor sample, of saliva, was positive for HHV-8. Sequencing showed 5% divergence among HHV-8 strains. The data suggest that saliva may be more important than semen or stool in the sexual transmission of HHV-8. The relatively high prevalence of HHV-8 in PBMC raises the question as to why there is no evidence for bloodborne virus transmission.

Epitope mapping of HTLV envelope seroreactivity in LGL leukaemia.

Sera from approximately 50% of patients with large granular lymphocyte (LGL) leukaemia react with a recombinant human T-cell leukaemia/lymphoma virus (HTLV) transmembrane envelope protein, p21e. Two immunodominant epitopes within env p21e have been defined by reactivity against recombinant proteins GD21 and BA21. In this study sera from 41 patients with LGL leukaemia were examined for reactivity against these recombinant HTLV env proteins. Overall, 21/41 (51%) sera reacted to p21e. Only two sera reacted to GD21. The predominant immunoreactivity against p21e was directed against the BA21 epitope, with 19/41 (46%) sera being BA21 positive. Seroconversion to BA21 protein was also documented. PCR analyses confirmed the low incidence of protypical HTLV sequences (2/41, 5%). These data document an association between BA21 seroreactivity and LGL leukaemia. This finding raises the possibility that such BA21 seroreactivity could be due to cross-reactivity to a cellular or retroviral antigen sharing some amino acid homology with the transmembrane glycoprotein of HTLV.

Large granular lymphocyte leukaemia occurring after renal transplantation.

Post-transplantation lymphoproliferative disorders (PTLD) are a clinicopathologically heterogeneous group of lymphoid proliferations. The majority are of B-cell origin and associated with Epstein-Barr virus (EBV) infection. In contrast, the development of T-cell PTLD is much less common and EBV does not appear to be involved in pathogenesis. In this report we describe three patients who developed large granular lymphocyte (LGL) leukaemia after renal transplantation. These patients had clonal expansion of CD3+, CD8+, CD57+, CD56- LGL. We were unable to detect CMV antigen or find evidence for EBV or human T-cell leukaemia/lymphoma virus genome in the LGL from these patients. These data show that LGL leukaemia should be included as one of the types of T-cell proliferations which can occur post transplant.

Expansion of clonotypic T-cell populations in the peripheral blood of asymptomatic Gran Chaco Amerindians infected with HTLV-IIB.

Peripheral blood mononuclear cells from asymptomatic HTLV-II-infected and uninfected Gran Chaco Amerindians were analyzed using polymerase chain reaction (PCR) for expansions of T-cell receptor (TCR) V-beta gene clonotypes. Analyses were performed using primer pairs designed to identify expanded T-cell familial clonotypes based on their unique TCR beta gene rearrangements. Of the 30 HTLV-IIB-positive samples tested, five showed evidence of V-beta clonotypic T-cell expansion. Of the five expansions, two were monoclonotypic and the remaining three were oligoclonotypic. In comparison, 30 HTLV-II-negative Amerindians showed no evidence of clonotypic T-cell expansion. Amplified DNA from one of the monoclonotypic samples was subsequently cloned and sequenced and was found to have uniform variable/ diversity/joining sequences confirming its unique monoclonal T-cell expansion. This method of detecting clonal TCR beta gene rearrangements has the advantage over traditional Southern blot techniques of being more sensitive and specific even with suboptimal specimens. The prognostic significance of clonotypic T-cell expansion in a group such as the HTLV-II-infected Gran Chaco Amerindians remains to be determined.

Endemic infection with HTLV-IIB in Venezuelan Indians: molecular characterization.

The peripheral blood of 41 Yaruro and Guahibo Indians from Venezuela was examined for HTLV antibodies and DNA. Twenty-five samples (61%) were found to be infected with HTLV-IIB. The sensitivities of the serologic and DNA polymerase chain reaction (PCR) analyses were 80% and 96%, respectively. Epidemiologic studies supported both sexual and perinatal transmission of the virus. Sequence analyses of the HTLV-IIB strains from these Indians indicate that they are unique relative to HTLV-II detected in other groups of humans. HTLV-IIB-G2 isolated from a Guahibo Indian is the most divergent HTLV-IIB strain relative to the prototype HTLV-II NRA.