Increased level of superoxide dismutase (SOD) activity in larvae of Chironomus ramosus (Diptera: Chironomidae) subjected to ionizing radiation.

A battery of enzymes from the eukaryotic antioxidant defense system was measured in salivary gland and in whole body extract of fourth instar larvae of Chironomus ramosus with an objective of finding any clue for the dipteran insect's capacity to tolerate heavy doses of ionizing radiation. Levels of activity of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSH-Px) were quantified in 30 days old larvae exposed to LD(20) dose of gamma radiation. Compared to controls, activity of Cu,Zn-SOD increased 3 to 4 fold and catalase 2 fold in response to ionizing radiation stress, while activities of GR and GSH-Px enzymes were decreased. Among the other SOD isoenzymes, our results showed comparable levels of Mn-SOD and Cu,Zn-SOD activity in control and irradiated groups of larvae. The increase in levels of the Cu,Zn-SOD isoenzyme was also confirmed by Western blot and zymography supported by densitometric quantification. No evidence of Fe-SOD was found in C. ramosus larvae. These findings could help to explain the persistence of natural populations of Chironomus in radioactively contaminated regions.

Activation of HHV-6 in lymphoproliferative disorders: a polymerase chain reaction-based study.

HHV-6 is a latent herpes virus persisting throughout the adult life of the infected host in an integrated form and is often activated in immunocompromised situations. Detection of HHV-6 DNA in the plasma of an individual indicates the presence of active viral replication in the host. Because lymphomas are known to be associated frequently with host immunosupression, we studied activation of HHV-6 in 98 patients diagnosed with Hodgkin's disease (HD) or non-Hodgkin's lymphoma (NHL). HHV-6 activation was documented in 34% of cases of non-Hodgkin's lymphoma and 39% of those of Hodgkin's disease; however, no correlation of activation status with pathological types of Hodgkin's disease and between copy numbers in peripheral blood mononuclear cell DNA and the corresponding plasma DNA was noticeable.

Perinatally cotransmitted human herpesvirus 6 is activated in children born with human immunodeficiency virus infection.

OBJECTIVE: To evaluate the mother-to-child transmission profile of human immunodeficiency virus (HIV) as well as human herpesvirus 6 (HHV-6) and to examine active replication of HHV-6 in the HIV-infected mothers and their newborns. STUDY DESIGN/METHODS: This polymerase chain reaction (PCR)-based detection was done using DNA from peripheral blood mononuclear cells (PBMCs) and milk cells from the mothers, PBMC from the newborns, and DNA derived from plasma and cell-free milk fluid from mothers and plasma of the newborns. None of the mothers received antiretroviral treatment. RESULTS: HIV was transmitted to 50% newborns and, of 36 total mothers, 8 had actively replicating HHV-6 detectable in their plasma and 2 also had it in the lactosera. Among the neonates. HHV-6 was found in the PBMC DNA of seven and in the plasma fractions of five, the latter five newborns were all HIV-infected at birth. CONCLUSION: Perinatally cotransmitted HHV-6 was always activated in the neonates who were born with HIV infection. Also, HHV-6 can be detected in the milk cells and the activated virus may be present in the lactosera of some of these HlV-infected mothers.

Unique organization of the CTX genetic element in Vibrio cholerae O139 strains which reemerged in Calcutta, India, in September 1996.

We studied the restriction fragment length polymorphism of the rRNA gene and CTX genetic element in Vibrio cholerae O139 Bengal, which resurged in Calcutta in September 1996 after a gap of 32 months. While the strains from this resurgence were indistinguishable from the earlier strains by ribotyping, the structure of the CTX genetic element present in the current O139 strains was found to be unconventional.

Regulation of follicular maturation by human ovarian follicular fluid peptide.

Follicular development, ovulation and luteal function are controlled by gonadotrophins. However, recent evidence indicates that local factors are also responsible for the regulation of folliculogenesis. In addition to their endocrine action on pituitary gonadotrophins, inhibin, activin and follistatin also have a paracrine role in follicular maturation. An ovarian follicular fluid peptide (OFFP) has been identified from sheep and humans. Purification of OFFP has been achieved by ultrafiltration and gel chromatography with further purification by fast performance liquid chromatography and reversed phase-high pressure liquid chromatography. OFFP is a small (< 5 kDa) peptide that competes with FSH in binding to granulosa cells in vitro and inhibits progesterone secretion from granulosa cells in culture. Immunohistochemical localization revealed the presence of OFFP mainly in granulosa cells of ovarian follicles. Furthermore, the peptide caused apoptosis in granulosa cells and induced follicular atresia. OFFP may act indirectly on oocytes via its effect on granulosa cells. The peptide from ovarian follicular fluid appears to have an important autocrine or paracrine role in the regulation of folliculogenesis.

Only late, nonmitotic stages of granulocyte differentiation in 32Dcl3 cells are blocked by ectopic expression of murine c-myb and its truncated forms.

In murine leukemia virus-induced myeloid leukemias, insertional mutagenesis of the c-myb locus has been shown to occur frequently. Proto-oncogene activation is achieved in most leukemias by integration of murine leukemia virus upstream of exons 3 or 4 or by integration into exon 9 with consequent truncation of the protein. The present study investigates the effect of ectopic expression of full-length c-myb or c-myb containing amino- or carboxyl-terminal truncations (minus 47 and 248 amino acids, respectively) on granulocyte differentiation in vitro. Recombinant myb retroviruses were used to infect an interleukin 3-dependent progenitor cell line, 32Dcl3, which undergoes terminal differentiation to mature neutrophilic granulocytes in the presence of granulocyte colony-stimulating factor. Overexpression of c-myb did not abrogate the interleukin 3 dependency of the parental cell line. However, cells expressing all forms of c-myb were blocked at an intermediate stage of granulocyte differentiation and continued to proliferate in the presence of granulocyte colony-stimulating factor. After 14 days in medium with granulocyte colony-stimulating factor, myb-expressing cultures predominantly consisted of promyelocytes with some myelocytes and almost undetectable numbers of neutrophilic granulocytes. This suggested that early stages of granulocyte differentiation were not inhibited, a finding that was further supported by the induction of myeloperoxidase, a biochemical marker of promyelocytes. Interestingly, the expression of lactoferrin, known to be a marker of late stages of granulocyte differentiation, was completely inhibited in the cells infected with myb viruses. It was concluded that c-myb expression blocked granulocyte differentiation to the terminal mitotic stages and that deletion of the NH2-terminal 47 amino acids and/or the COOH-terminal 248 amino acids of c-myb neither enhanced nor diminished this effect.

Different abilities of Friend murine leukemia virus (MuLV) and Moloney MuLV to induce promonocytic leukemia are due to determinants in both psi-gag-PR and env regions.

Moloney murine leukemia virus (M-MuLV) is capable of inducing promonocytic leukemia in 50% of adult BALB/c mice that have received peritoneal injections of pristane, but Friend MuLV strain 57 (F-MuLV) is nonleukemogenic under similar conditions. It was shown earlier that these differences could not be mapped to the U3 region of the virus long terminal repeat, indicating the probable influence of structural genes and/or R-U5 sequences. In this study, reciprocal chimeras containing exchanged structural genes and R-U5 sequences from these two closely related viruses were analyzed for differences in ability to induce disease. Results showed that two regions of F-MuLV, psi-gag-PR and env, when substituted for those of M-MuLV were dramatically disease attenuating. The 5'-most region, which is widely distributed, overlaps with the 5' end of the env intron and includes the RNA packaging region, psi, the entire gag coding region, and the viral protease coding region (PR) of pol. It was also found that reciprocal constructs having substitutions of both of these regions of M-MuLV in an F-MuLV background allowed full reestablishment of promonocytic leukemia. These leukemias were positive for c-myb rearrangements which are characteristic of M-MuLV-induced promonocytic leukemias. Neither region alone, however, was sufficient to produce disease with a greater incidence than 13%. Further studies demonstrated that the inability of viruses with psi, gag, PR, or env sequences from F-MuLV to induce leukemia in this model system was not due to their inability to replicate in hematopoietic tissue, to integrate into the c-myb locus early on after infection in vivo, or to express gag-myb mRNA characteristic of M-MuLV-induced preleukemic cells and acute leukemia.

Nucleotide sequence analysis of HTLV-I isolated from cerebrospinal fluid of a patient with TSP/HAM: comparison to other HTLV-I isolates.

Human T-cell leukemia virus type I (HTLV-I) has been associated with adult T-cell leukemia/lymphoma and the chronic neurologic disorder tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). To study the genetic structure of the virus associated with TSP/HAM, we have obtained and sequenced a partial genomic clone from an HTLV-I-positive cell line established from cerebrospinal fluid (CSF) of a Jamaican patient with TSP/HAM. This clone consisted of a 4.3-kb viral sequence containing the 5' long terminal repeat (LTR), gag, and N-terminal portion of the pol gene, with an overall 1.3% sequence variation resulting from mostly nucleotide substitutions, as compared to the prototype HTLV-I ATK-1. The gag and pol regions showed only 1.4% and 1.2% nucleotide variations, respectively. However, the U3 region of the LTR showed the highest sequence variation (3.6%), where several changes appear to be common among certain TSP/HAM isolates. Several of these changes reside within the 21-bp boundaries and the Tax-responsive element. It would be important to determine if the observed changes are sufficient to cause neurologic disorders similar to the murine leukemia virus system or simply reflect the divergent pool of HTLV-I from different geographic locations. At this time, we cannot rule out the possibility that the observed changes have either direct or indirect significance for the HTLV-I pathogenesis in TSP/HAM.

New sites of proviral integration associated with murine promonocytic leukemias and evidence for alternate modes of c-myb activation.

Murine promonocytic leukemias involving insertional mutagenesis of the c-myb locus can be induced by replication-competent retroviruses. In previously studied promonocytic leukemic cells induced by Moloney murine leukemia virus (called MML), the provirus has been invariably integrated upstream of exons 3 or 4 and the leukemic cells expressed aberrant RNAs with fused virus-myb sequences. Furthermore, Myb expressed by these cells has been shown to be truncated by 47 or 71 amino acids. The present report examines the mechanisms of myb activation in leukemias induced by two other retroviruses, amphotropic virus 4070A and Friend strain FB29 (the leukemias are called AMPH-ML and FB-ML, respectively). This study revealed two additional c-myb proviral insertion sites in these promonocytic leukemias. One FB-ML had a proviral integration in exon 9, and expressed a C-terminally truncated Myb protein of 47 kDa similar to that previously demonstrated to be expressed in the myelomonocytic cell lines NFS60 and VFL-2. However, a sequence of reverse-transcribed and amplified RNA from this leukemia demonstrated that the truncation involved a loss of 248 amino acids compared with a loss of 240 amino acids in the myelomonocytic cell lines. Another leukemia had a provirus integrated in the 5' end of c-myb upstream of exon 2 (in the first intron) and produced a Myb protein that was indistinguishable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from normal Myb. This latter leukemia (FB-ML R1-4-10) expressed Myb with the smallest N-terminal truncation observed so far in promonocytic leukemias; translation begins at an ATG within c-myb exon 2, leading to loss of only 20 amino acids from the N terminus. Unlike the proteins produced in Moloney murine leukemia virus-induced promonocytic leukemias (MML) that have larger truncations, this protein has an intact DNA binding region and does not contain N-terminal amino acids encoded by gag. However, this protein is similar to all N-terminally truncated Mybs so far studied, in that the truncation resulted in deletion of a casein kinase II phosphorylation site which has been proposed to be involved in regulation of DNA binding.

Conservative mutations in the putative metal-binding region of human immunodeficiency virus tat disrupt virus replication.

The tat trans-activator proteins of the primate immunodeficiency viruses contain a highly conserved cysteine-rich domain. In human immunodeficiency virus type 1 tat there are seven cysteines located between residues 22 and 37 that are thought to form a metal-nucleic acid-binding structure. Most of the previous mutagenesis studies had demonstrated that these residues are essential for tat activity and virus expression. Here we show that potentially conserved cysteine-histidine substitutions within the proposed tetrahedral structure still eliminate tat activity and virus expression. Consistent with previous studies, one cysteine-to-histidine mutation (amino acid 31) had little effect on trans-activation. We have studied the functional properties, stability and subcellular localization of several tat protein mutants. Most of the mutants are stable and properly localized to the nucleus and/or nucleolus. However, cysteine-to-glycine at position 34 affected tat stability. Our studies with the histidine mutants suggest that tat does not assume the prototype "zinc finger" structure for metal binding.

Immunoreactivity of lymphocytes from draining lymph nodes, peripheral blood and tumor infiltrates from oral cancer patients.

Lymphocytes from metastatic (met) and nonmetastatic (non-met) regional lymph nodes, LNL peripheral blood (PBL) and tumor infiltrating lymphocytes (TIL) of patients with squamous cell carcinoma of the oral cavity and healthy donors were investigated for CD3, CD4, CD8 and HNK-1 phenotypes, Natural Killer (NK) cell Activity, Antibody Dependent Cellular Cytotoxicity (ADCC) and proliferative response to mitogen (PHA). Modulation of NK cytotoxicity with recombinant Interferon alpha (IFN-alpha) was also investigated in some cases. Lymphocytes from met and non-met lymph nodes showed no variation in the percentages of CD3+, CD4+ and CD8+ cells, when compared with each other and with PBL of oral cancer patients. TIL showed significantly less proportion of CD3+ and CD4+ cells. The percentage of HNK-1+ cells was significantly lower in LNL and TIL when compared to PBL of oral cancer patients. The mitogen responses of met and non-met LNL were comparable to each other and better than that of PBL from the same patients, while, TIL showed significant impairment in mitogen responses. The NK cytotoxicity and ADCC of PBL from oral cancer patients were comparable to healthy donors which could be augmented by rIFN alpha. LNL and TIL showed almost negligible NK and ADCC activities and NK activity could not be modulated by rIFN alpha. The results thus demonstrate that in oral cancer patients, lymphocytes from three compartments viz. PBL, LNL and TIL showed differential effector functions. The metastatic status of LN did not affect the immunoreactivity of LNL.

Natural killer and lymphokine-activated killer cell-mediated cytotoxicity in patients with oral cancer.

Peripheral blood lymphocytes (PBL) from untreated and treated oral cancer patients, lymph node lymphocytes (LNL) from metastatic (met) and nonmetastatic (non-met) lymph nodes, and tumor infiltrating lymphocytes (TIL) were tested for natural killer (NK) and lymphokine activated killer (LAK) cell cytotoxicity using appropriate targets in a short-term chromium release assay. The results showed that while both NK and LAK functions of PBL from oral cancer patients were comparable to those of normal healthy donors, the NK activity of metastatic and nonmetastatic LNL and TIL was highly compromised. On the other hand, potent LAK activity could be generated from all three lymphoid populations. Individual patients showing low NK activity displayed good LAK cytotoxicity, indicating that endogenous cells with low NK potential have adequate ability to respond to interleukin 2 (IL-2). LAK activity tested on autologous tumour targets revealed that TIL were the best source of LAK cells. followed by PBL and LNL.

Effect of exogenous interleukins on in vitro responses of T lymphocytes from patients with Hodgkin disease.

PHA-induced peripheral blood lymphocyte (PBL) proliferation and T-cell colony formation in soft agar was studied in the presence of interleukin-2 (IL-2) containing medium in untreated Hodgkin disease (HD) patients and normal controls. The proliferative response of HD PBL was potentiated in presence of IL-2-containing medium in 11 of 20 cases studied, and there was marginal increase or inhibition of PHA response in 16 normal lymphocyte samples. Lymphocytes from patients in advanced stages of the disease, and those who were initially hyporesponsive responded better to suboptimal doses of PHA in the presence of exogenous IL-2. There was no improvement in the colony forming capacity of T lymphocytes from HD PBL even in the presence of IL-2-containing medium, whereas normal T-cell colony formation was augmented significantly both in number and in size of colonies.

Detection of circulating immune complexes in patients with squamous cell carcinoma of the oral cavity.

Circulating immune complexes (CICs) have been quantitated from the sera of untreated oral cancer patients and those treated with radiation or surgery using C1q binding assay in terms of percent binding activity (C1q BA) and microgram/ml equivalent of aggregated human gamma globulin (AHG). Sera from a total of 108 oral cancer patients and 47 normal healthy donors were evaluated for CICs. Out of these, 48 patients were tested before treatment and 60 were tested 6 months to 1 yr after treatment. Levels of CICs were elevated in 70.8% patients before treatment (mean % C1q BA 29.6 +/- 2.2) when compared to healthy controls (4.2% positive, mean % C1q BA 9.0 +/- 0.8). Treated patients showing no evidence of the disease had reduced CIC levels, only 11.7% patients showing C1q BA at the level of 13.2 +/- 1.4. On the other hand, treated patients showing recurrence of the disease had much higher CIC levels (mean % C1q BA 42.8 +/- 3.5, 92.3% positive) even higher than the untreated patients. Status of CIC levels as well as recurrence rate in patients treated with either radiation or surgery were comparable. One out of 4 individuals with premalignant changes such as oral leukoplakia showed elevated levels of CICs.

Cell-mediated immune status in patients with squamous cell carcinoma of the oral cavity.

Sixteen untreated patients with squamous cell carcinoma of the oral cavity were tested for in vitro immune status in comparison with the normal healthy donors. The parameters investigated were total leukocyte and lymphocyte counts, percentages and absolute counts of T- and B-cells in circulation, subsets of T-cells identified by the Fc receptors, phytohemagglutinin (PHA), and mixed lymphocyte culture (MLC) responses, natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities, and circulating immune complexes (CICs). Eight of these patients were retested 3 to 6 months after surgery. The results showed that there was an increase in leukocyte and lymphocyte counts, an increase in the percentage and absolute number of B-lymphocytes, an increase in the percentage of T-gamma cells, suboptimal PHA and MLC responses, normal NK and ADCC activities, and increased levels of CICs in untreated oral cancer patients. In the postoperative stage, except for a reduction in leukocyte and lymphocyte counts, other abnormalities remained unchanged. The CICs in treated patients correlated with the tumor load in that in three patients showing recurrence, the CIC level remained elevated, whereas in patients without evidence of the disease the CIC level was either low or comparable to the upper normal limits.

Impairment of T lymphocyte colony formation in Hodgkin's disease: effect of soluble inhibitory factors on normal T lymphocyte colony formation potential.

PHA-induced T lymphocyte colonies from peripheral blood mononuclear cells of untreated Hodgkin's disease (HD) patients and normal healthy donors were assayed by one-step stimulation in microagar capillary cultures. A significant depression in the T cell colony formation was observed in HD patients in comparison with normal healthy donors. Clinical staging of the disease had no influence on this abnormality. Preincubation of HD lymphocytes for 24 h in tissue culture medium did not produce any appreciable recovery in the colony formation potential. However, in the presence of 24-hour culture supernatants of HD mononuclear cells, there was significant inhibition in the colony formation by lymphocytes obtained from normal healthy donors. Significance of these observations is discussed.