Donor demographics and factors affecting corneal utilisation in Eye Bank of North India.

INTRODUCTION: Nearly 6.8 million people in India have vision less than 6/60 in at least one eye due to corneal diseases; of these, about a million had bilateral involvement. PURPOSE: To identify the challenges faced; the trends in collection, storage and utilisation of corneal tissues in an eye bank in north India. MATERIALS AND METHODS: The past records of Eye Bank linked to a tertiary hospital in northern India were analysed from November'1999 to October'2015 with respect to number of eye donations per year, donor demographics and utilisation of corneal tissues. RESULTS: The number of donations during the first 6 years were 100, 279 in the next 5 years and 473 in the last 5 years. The mean donor age was 63.2 ± 19.5 years. The percentage of donors less than 30, 31-60 and more than 60 years was 10%, 28% and 62%. Forty-two percent donations were from the hospital. The average time between the death and enucleation was 4.74 ± 5.31 hours. The percentage of corneas used in the donor age groups less than 30, 31-60 and above 60 years was 61.9%, 61.6% and 53.8%, respectively. The usability rate of the corneas from home and hospital was 63.7% and 55.3%, respectively. CONCLUSIONS: The eye bank had a lukewarm response in the beginning, but gained momentum with time. The myths and beliefs prevalent in our society deter people from donating eyes freely. Each eye bank needs to individualise its problems and find solutions for adequate procurement and utilisation of tissue.

T-cell growth factor gene: lack of expression in human T-cell leukemia-lymphoma virus-infected cells.

Activated mature T cells require T-cell growth factor (TCGF) for continuous proliferation. However, many mature T cells infected with human T-cell leukemia-lymphoma virus grow independently of exogenously added TCGF. It is now reported that cells infected with this virus also lack detectable TCGF messenger RNA (less than one copy per cell) and thus do not produce their own growth factor. The results apparently rule out an autostimulation mechanism of growth control.

Trans-activator gene of human T-lymphotropic virus type III (HTLV-III).

Human T-lymphotropic virus type III (HTLV-III) encodes a trans-acting factor that activates the expression of genes linked to the HTLV-III long terminal repeat. By functional mapping of complementary DNA transcripts of viral messenger RNA's the major functional domain of the gene encoding this factor was localized to a region immediately before the env gene of the virus, a region previously thought to be noncoding. This newly identified gene consists of three exons, and its transcription into messenger RNA involves two splicing events bringing together sequences from the 5' part (287 base pairs), middle (268 base pairs), and 3'part (1258 base pairs) of the HTLV-III genome. A similar messenger RNA with a truncated second exon (70 base pairs) does not encode a trans-acting function. It is proposed that this second messenger RNA is the transcript of a gene (3'-orf) located after the env gene. Messenger RNA's were also identified for the env and gag-pol genes of HTLV-III.

Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells.

Evidence suggests that CD8+ T lymphocytes are involved in the control of human immunodeficiency virus (HIV) infection in vivo, either by cytolytic mechanisms or by the release of HIV-suppressive factors (HIV-SF). The chemokines RANTES, MIP-1 alpha, and MIP-1 beta were identified as the major HIV-SF produced by CD8+ T cells. Two active proteins purified from the culture supernatant of an immortalized CD8+ T cell clone revealed sequence identity with human RANTES and MIP-1 alpha. RANTES, MIP-1 alpha, and MIP-1 beta were released by both immortalized and primary CD8+ T cells. HIV-SF activity produced by these cells was completely blocked by a combination of neutralizing antibodies against RANTES, MIP-1 alpha, and MIP-1 beta. Recombinant human RANTES, MIP-1 alpha, and MIP-1 beta induced a dose-dependent inhibition of different strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV). These data may have relevance for the prevention and therapy of AIDS.

Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome.

The human T-cell leukemia (lymphotropic) virus type III (HTLV-III) appears to be central to the causation of the acquired immune deficiency syndrome (AIDS). Two full-length integrated proviral DNA forms of HTLV-III have now been cloned and analyzed, and DNA sequences of the virus in cell lines and fresh tissues from patients with AIDS or AIDS-related complex (ARC) have been characterized. The results revealed that (i) HTLV-III is an exogenous human retrovirus, approximately 10 kilobases in length, that lacks nucleic acid sequences derived from normal human DNA; (ii) HTLV-III, unlike HTLV types I and II, shows substantial diversity in its genomic restriction enzyme cleavage pattern; (iii) HTLV-III persists in substantial amounts in cells as unintegrated linear DNA, an uncommon property that has been linked to the cytopathic effects of certain animal retroviruses; and (iv) HTLV-III viral DNA can be detected in low levels in fresh (primary) lymphoid tissue of a minority of patients with AIDS or ARC but appears not to be present in Kaposi's sarcoma tissue. These findings have important implications concerning the biological properties of HTLV-III and the pathophysiology of AIDS and Kaposi's sarcoma.

Homology of genome of AIDS-associated virus with genomes of human T-cell leukemia viruses.

A T lymphotropic virus found in patients with the acquired immune deficiency syndrome (AIDS) or lymphadenopathy syndrome has been postulated to be the cause of AIDS. Immunological analysis of this retrovirus and its biological properties suggest that it is a member of the family of human T-lymphotropic retroviruses known as HTLV. Accordingly, it has been named HTLV-III. In the present report it is shown by nucleic acid hybridization that sequences of the genome of HTLV-III are homologous to the structural genes (gag, pol, and env) of both HTLV-I and HTLV-II and to a potential coding region called pX located between the env gene and the long terminal repeating sequence that is unique to the HTLV family of retroviruses.

Usability of donor corneas harvested from the deceased having septicaemia or malignancy.

CONTEXT: There is a wide gap between supply and demand in relation to healthy corneal grafts. Specific contraindications like infection and malignancy lead to non-usage of many grafts, despite the fact that deeming graft unhealthyness for these two contraindications is debatable. AIMS: This study was conceptualized to assess if corneas donated from the deceased with septicaemia or malignancy can be deemed fit for implantation. SETTINGS AND DESIGN: Retrospective histopathological and microbiological analysis of cadaveric donor corneas. METHODS: A total of 76 donor corneas from 38 patients rejected for corneal transplantation in view of patient having septicaemia or malignancy were analysed for pathological and microbiological workup, to look for dissemination of disease within corneal tissue. Pathology workup included gross and microscopic histopathological evaluation of tissue. Microbiology workup included Grams stain and KOH with calcofluor mount, culture in blood agar, chocolate agar, Sabourauds dextrose agar and Mc Conkeys broth. RESULTS: A total of 46 donor corneas of 23 septicaemia patients when evaluated showed presence of culture positive infection in 18 patients (78.2%). Histopathological examination done for 30 donor corneas from 15 cancer patients did not reveal presence of tumour cells in the specimen. Corneas of two of cancer patients having septicaemia revealed growth on cultures. CONCLUSIONS: Corneal tissues harvested from septicaemia donors showed significantly higher incidence of corneal contamination, confirming their unsuitability for usage. However, there was no incidence of tumour transmission in corneal tissues of the patients with malignancies, suggesting that they can be considered for ophthalmic purpose.

Transcriptional regulation of a tumor promoter and mitogen-inducible gene in human lymphocytes.

Tumor-promoting phorbol esters affect a variety of cellular functions which may underlie tumor promotion. We isolated from human lymphocytes a cDNA clone whose gene is inducible by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate as well as by the T-cell mitogen phytohemagglutinin. Nuclear transcription experiments suggested that this induction is primarily caused by the increased transcription of the gene. It is interesting that this gene is expressed constitutively in human T-cell leukemia virus-infected mature T cells. The results support the notion that 12-O-tetradecanoyl-phorbol-13-acetate can affect cellular functions by causing transcriptional activation of specific genes.

Comparison of the sequence complexity and abundance frequency of polyadenylated messenger RNA in human breast and prostate carcinoma cells.

The sequence complexity and frequency distribution of polyadenylated [poly(A)+] messenger RNA (mRNA) in human breast and prostate carcinoma cells were compared by the kinetics of homologous and heterologous mRNA:complementary DNA hybridization. The apparent total sequence complexity of human breast cell poly(A)+ RNA was estimated to be about 47,300 kilobases (kb). It appeared to be distributed in three abundance classes comprising about 14 (32 kb), 43 (2,000 kb), and 43% (45,270 kb) of the total. The poly(A)+ mRNA of human prostate cells also appeared to be organized in three abundance classes: the highest-abundance class comprising about 15% of the total with an approximate complexity of 88 kb; an intermediate-abundance class comprising about 32% of the total with a complexity of about 2,330 kb; and a low-abundance class which was about 53% of the total and had an estimated complexity of 66,840 kb. The results of heterologous hybridization reaction suggested that about 75 to 80% of the poly(A)+ mRNA sequences present in human breast cells were also present in human prostate cells. Similarly, about 75 to 80% of the sequences in human prostate cells appeared to be present in human breast cells. The sequences held in common by these two cell types appeared to be present in roughly similar relative abundance. The hybridization of kinetically fractionated abundant complementary DNA of breast or prostate cells with homologous and heterologous poly(A)+ mRNA suggested that the abundant sequences of one cell type (e.g., breast) were present in reduced abundance in the other cell type (e.g., prostate) but only by a factor of 2 to 4. Nearly all of the abundant sequences of one cell type appeared to be present in the second cell type.

Changes in retrovirus expression in mouse mammary tumor cells in culture.

Mouse mammary tumor cells (Mm5Mt/c1) produced mouse mammary tumor virus (MMTV) (type B retrovirus) at early passages in culture. They apparently produced predominently type C retrovirus(es) at late passages in culture. The decline in MMTV production during passage was not accompanied by an apparent decrease in the synthesis or accumulation of intracellular MMTV-specific RNA. As judged by the concentration of RNA (mol/titer) x half-time (sec) of hybridization of cellular RNA with MMTV complementary DNA, the intracellular concentration MMTV-specific RNA did not change significantly during passage in culture. Since late-passage RNA, particularly polysomal poly(adenylic acid)-containing RNA, yielded lower apparent maximum hybridization of MMTV complementary DNA as compared with early-passage RNA, it was possible that the complexity or base sequence of MMTV-specific RNA in late-passage cultures was not identical with that in early-passage cultures. The data on thermal transitions of hybrids were in accord with this possibility. In contrast, the steady-state level of type C virus-specific RNA in late-passage cultures was orders of magnitude higher than that in early-passage cultures. The production of type C retrovirus(es) at later passages was apparently accompanied by increased transcription of type c retrovirus DNA.

Search for retrovirus-like particles in human breast cancer cells in culture.

We have recently established four new human breast cancer cell lines that were characterized as being of human mammary origin. We examined these cell lines for particles morphologically resembling retroviruses by electron microscopy, for extracellular and intracellular particles containing high-molecular-weight RNA and RNA-directed DNA polymerase by biochemical assays, and for mouse mammary tumor virus (MMTV)-related sequences in the cell genomes by molecular hybridization. An extensive search for budding particles by thin-section electron microscopy of cells did not provide evidence for retrovirus-like particles. Similarly, 1000- to 2000-fold concentrated samples of medium harvested from 10(8) cells did not contain particles of a density of 1.14 to 1.16 g/ml containing RNA-directed DNA polymerase. Compared with DNA polymerase activity of MMTV, and taking into account the particle weight and protein content of retroviruses, we estimate that, if these cells produce retrovirus-like particles, this production would be less than 1.6 particles/cell every 24 to 72 hr. The hybridization of cell DNA with MMTV complementary DNA also did not show detectable amounts of virus-related sequences in the cell genome. Analysis of the hybridization results suggested that, if the human breast cells contained MMTV-related sequences, they must be present in less than one copy per 100 cells. Thus, we have obtained no convincing evidence for the presence of retrovirus-like particles or subviral components in these cells. It is of course possible that these cells contain virus information but at levels below the sensitivity of our assay procedures.

Effect of Variable Oxidation States of Vanadium on the Structural, Optical, and Dielectric Properties of B2O3-Li2O-ZnO-V2O5 Glasses.

In the present study, the effect of variable vanadium oxidation states on the structural, optical, and dielectric properties of vanadium oxide containing lithium borate glasses has been investigated. Electron paramagnetic resonance studies indicate that vanadium in these glasses is mostly in the V4+ state, having a tetragonal symmetry. As the glass composition of V2O5 increases, tetragonality also increases at the cost of octahedral symmetry. The photoluminescence (PL) spectra of these glasses are dominated by zinc oxide transition, whereas the peaks pertaining to the vanadyl group are not visible in the PL spectra. The optical absorption spectra show a single wide absorption band, which is attributed to V4+ ions in these glasses. The ac conductivity of the glasses increases with an increase in vanadium content. The highest electrical conductivity observed is ∼10-5 S cm-1 at 250 °C for the glass with 2.5 mol % V2O5. Electrical conductivity is dominated by electron conduction, as indicated by the activation energy calculation.

Inhibition of HIV-1 expression by HIV-2.

HIV-1 and HIV-2 are co-endemic in certain geographic areas. HIV-2 is more weakly pathogenic than HIV-1, and progression to AIDS occurs less frequently and over a longer period of time. Recent epidemiologic studies suggest that individuals infected with HIV-2 have a lower risk of HIV-1 infection. Both immune mechanisms and various modes of viral interference have been proposed to account for these results. Our findings, described in this paper, suggest that HIV-2 inhibits HIV-1 replication. To study the molecular interactions between HIV-1 and HIV-2, proviral clones were transfected alone or in combination into the human T cell line CEM. LTR-CAT indicator constructs were included for the purpose of monitoring viral promoter activity. Viral replication in transfected cells was monitored by p24 antigen capture assay of cell culture supernatants and Western blot analysis of cell extracts. HIV-2 inhibited HIV-1 replication as determined by intracellular and extracellular p24 antigen levels. Similar results were obtained with simultaneous virus infection using HIV-1 and HIV-2, rather than transfections of proviral DNA. Using cotransfection of HIV-1 and HIV-2 LTR indicator gene constructs, the mechanism of inhibition was found to be suppression of the HIV-1 LTR by HIV-2. The inhibitory effect of HIV-2 is not due to Tat-2, but appears to discriminate between the HIV-1 and HIV-2 LTRs based on differences in the Tat activation response element, TAR. These results suggest both a molecular mechanism for HIV-2 interference with HIV-1 replication and a potential molecular approach to therapy.

Human immunodeficiency virus type 2 lentivirus vectors for gene transfer: expression and potential for helper virus-free packaging.

In addition to the long-term expression of the transgene provided by all retroviral vectors, lentiviruses present the opportunity to transduce nondividing cells and potentially achieve regulated expression. The development of lentiviral vectors requires the design of transfer vectors to ferry the transgene with efficient encapsidation of the transgene RNA and with full expression capability, and of a packaging vector to provide packaging machinery in trans but without helper virus production. For both vectors, a knowledge of packaging signal is required-the signal to be included in the transfer vector but excluded from the packaging vector. Among the human lentiviruses, human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2), we think HIV-2 is better suited for gene transfer than HIV-1. It is less pathogenic and thus safer during design and production; its desirable nuclear import and undesirable cell-cycle arrest functions are segregated on two separate genes. In HIV-1 infection, it is less likely to recombine with the resident HIV-1, and it may itself downregulate HIV-1 expression. Evidently, elements located both upstream and downstream of the splice donor site in the leader sequence participated in RNA encapsidation and these sequences appeared necessary and sufficient. Deletion of both sequence elements resulted in a dramatic curtailment of RNA encapsidation and helper virus production. This was accompanied by some but acceptable loss of gene expression capability. The helper virus-free phenotype and expression capability of the double mutant was maintained upon replacement of its 3' long terminal repeat with a minigene cassette containing a transcriptional termination signal and a drug resistance marker gene. Deletion of the splice donor site itself had a dramatic negative effect on gene expression, supporting the important role of this element in the life of RNA.

Human immunodeficiency virus type 2 (HIV-2): packaging signal and associated negative regulatory element.

Human immunodeficiency virus type 2 (HIV-2)-based retroviral vectors will have several desirable features as vehicles for gene therapy. These include target cell specificity, regulated expression, and attenuated cytopathicity. Such vectors require efficient packaging of RNA into retroviral particles which depends on a cis-acting sequence element called packaging signal or psi site. For most retroviruses, the principal part of this element is located between the major splice donor site and the gag initiator codon (AUG) in the leader sequence. The deletion of the corresponding region of HIV-2 did indeed cause a packaging defect; however, it did not abolish RNA encapsidation and viral infectivity. Additionally, deletions in this region resulted in an increase in intracellular viral RNA and extracellular p27 core antigen. However, only a fraction of the intracellular viral RNA was packaged into mature particles. These effects appeared to be sequence specific as deletion of the sequence elements upstream of the splice donor site did not result in increased viral RNA and proteins. A computer-assisted analysis of the leader sequence of viral RNA shows it to be rich in secondary structure, which was markedly altered in the deletion mutants. Thus, the leader sequence of HIV-2 between the splice donor site and the gag ATG has at least two regulatory functions: one positive, affecting encapsidation, and the other negative, regulating virus expression. Because there is only a limited sequence or structural homology between the corresponding region of HIV-1 and HIV-2, they are likely to differ in their pathways regulating packaging and gene expression.

D-penicillamine inhibits transactivation of human immunodeficiency virus type-1 (HIV-1) LTR by transactivator protein.

D-Penicillamine, an amino acid analogue of cysteine, has been shown to inhibit the transactivation of HIV-1 LTR by the transactivator protein, tat protein. The transactivation was studied in Jurkat cells co-transfected with plasmids containing HIV-LTR sequences fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and HIV tat gene. The expression of CAT activity was a measure of transactivation of LTR by the tat protein. Incubation of transfected Jurkat cells with D-penicillamine led to inhibition of CAT activity. This inhibition was found to be concentration-dependent; more than 90% inhibition of chloramphenicol acetylation was seen in extracts prepared from cultures incubated with 40 micrograms/ml of D-penicillamine. Earlier experiments have shown that D-penicillamine at 40 micrograms/ml can completely inhibit HIV-1 (HTLV-III B) replication in H9 cells [(1986) Drug Res. 36, 184-186]. These results suggest that inhibition of transactivation may be the molecular mechanism involved in the inhibition of HIV-1 replication by D-penicillamine.

Fusogenicity of mutant and chimeric proviruses derived from molecular clones of cytopathic and noncytopathic human immunodeficiency virus type 2.

Analysis of the phenotype of the molecular clones of cytopathic and fusogenic, noncytopathic and nonfusogenic, and chimeric proviruses of human immunodeficiency virus type 2 (HIV-2) suggests that the major determinant of the attenuated fusogenicity and cytopathicity of HIV-2 was located in the 3'-half of the genome, with envelope playing the more dominant role. However, no single linear domain within the envelope, including the major CD4 binding domain and fusogenic domain, was sufficient by itself for syncytia induction and cytopathic effects. Truncation of the transmembrane envelope glycoprotein downstream of the transmembrane region was not a major factor in this regard. However, truncation within the transmembrane region rendered the provirus replication incompetent. The regulatory genes (tat, rev) and auxiliary gene (nef) did not seem to play a critical role in determining HIV-2 fusogenicity in vitro. The results suggest the importance of the overall conformation of the envelope in the divergent phenotypes of HIV-2.

Human immunodeficiency virus type 2 (HIV-2) trans-activator (Tat): functional domains and the search for trans-dominant negative mutants.

Human immunodeficiency virus type 2 (HIV-2) trans-activator (Tat) is an important trans-regulator of viral gene expression. It differs from the related HIV-1 Tat in certain aspects of its structure and function. HIV-2 Tat is composed of 130 amino acids versus 86 amino acids for HIV-1 Tat. Apart from certain conserved regions, there is little homology between the two Tats. They also differ in their ability to trans-activate HIV-2 and HIV-1 long terminal repeat (LTR)-directed gene expression. As an aid to understanding its mechanism of action, the functional domains important for HIV-2 Tat trans-activation of HIV-2 and HIV-1 LTR-directed gene expression were investigated. Like HIV-1 Tat, HIV-2 Tat contains conserved cysteine- and arginine-rich domains important for its function. However, HIV-2 Tat differs from HIV-1 Tat in that about 20% of the HIV-2 Tat at the amino terminus was not essential for its trans-activation function while HIV-1 Tat amino terminus is reportedly a part of its activation domain. Similarly, about 30% of the protein at the carboxy terminus of HIV-2 Tat was not essential. A domain critical for HIV-2 Tat-mediated trans-activation was located just upstream of the cysteine-rich domain. This segment is predicted to adopt an alpha-helical conformation and also contains acidic amino acid residues; thus, it may resemble amphipathic helix-type activation domains found in some transcriptional factors. A region with predicted hydrophobic alpha-helical character located between the cysteine- and arginine-rich domains was also important for HIV-2 Tat function. HIV-2 Tat mutants that were analogs of HIV-1 Tat trans-dominant negative mutants did not display such a phenotype.

Human immunodeficiency virus type 2 (HIV-2) gene expression: downmodulation by sequence elements downstream of the transcriptional initiation site.

Human immunodeficiency virus type 2 (HIV-2) gene expression is downmodulated by sequence elements downstream of the transcriptional initiation site, corresponding to the U5 region of the long terminal repeat (LTR) and further downstream. This repression appeared to be related more to the length of the sequence intervening the transcriptional initiation site and the coding region than to a particular sequence content. The repressive effect of the downstream segment was not affected by HIV-2 and HIV-1 TAT or by the cytomegalovirus transactivator IE-2 gene. Nor was it affected by T-cell activation signals or by such cytokines as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interferon-gamma (IFN gamma), and interferon-alpha (IFN alpha). In contrast to HIV-1, HIV-2 LTR-directed gene expression was not modulated by TNF-alpha. A specific sequence element, located downstream of the TAR element in the R region, seemed to participate in modulation of gene expression. This element interacted with a nuclear protein with a mobility of about 26 kD. The repressive effect of the downstream sequence was to a certain extent cell type dependent, suggesting the involvement of cell type-specific factors. It was more effective in human lymphocytic CEM cells than in Jurkat cells. This may be relevant to the HIV-2 cell tropism (replication), latency, and virulence.

Human and simian immunodeficiency retroviruses: activation and differential transactivation of gene expression.

New West African human immunodeficiency viruses (HIV-2s) and simian immunodeficiency virus (SIV) contain functional transactivator (tat) gene and tat response elements. Their long terminal repeats (LTR) and tat genes are more related among themselves than to HIV-1 LTR and tat gene. The viral gene expression of HIV-2 as well as SIV can be stimulated by T cell activators, such as mitogens and phorbol esters. HIV-2 and SIV display a much broader transactivation response specificity than does HIV-1. The LTR-directed gene expression of HIV-2/SIV is not only transactivated by their own tat gene and by HIV-1 tat gene but also by factors in human T cell leukemia/lymphoma virus type I (HTLV-I) and simian virus 40 (SV40) infected cells, involving HTLV-I tat gene and SV40 T antigens, respectively. HIV-1 LTR-directed gene expression is much less transactivated by HIV-2/SIV tat genes and by factors in HTLV-I- and SV40-infected cells. Immune activation and heterologous transactivation of the LTR-directed gene expression may be relevant to the latency of virus infection and progression toward the acquired immunodeficiency syndrome (AIDS).