Biomedical subjects
R K Puri
Publications and source records attributed to R K Puri.
An ultrastructural study of in vivo interactions between lymphocytes and endothelial cells in the pathogenesis of the vascular leak syndrome induced by interleukin-2.
Lymphokine-activated killer (LAK) cells play a major role in the induction of the vascular leak syndrome (VLS). To understand the mechanism of this syndrome, the authors examined light and electron microscopic alterations in the lung, liver, spleen, kidney, and heart of mice in which VLS was produced by the administration of interleukin-2 (IL-2) (seven injections of 600,000 IU each for a period of 4 days). The results of these studies disclosed that considerable damage had been done to the endothelial cells that consisted of cytoplasmic edema, vacuoles, and myelin figures; in addition, there were frequent sites of transendothelial passage of lymphoid cells, probably IL-2-activated cells, that penetrated through their cytoplasm by means of "temporary migration pores" and accumulated in the perivascular spaces. The results of this study indicate that a direct in vivo interaction between IL-2-activated cells (probably LAK cells) and endothelium results in cytotoxicity to endothelial cells.
Correspondence Re: E. Gorelik et al., Effect of ultraviolet irradiation on MCA102 tumor cell immunogenicity and sensitivity to tumor necrosis factor. Cancer Res., 51: 1521-28, 1991.
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Expression of high-affinity interleukin 4 receptors on murine sarcoma cells and receptor-mediated cytotoxicity of tumor cells to chimeric protein between interleukin 4 and Pseudomonas exotoxin.
The presence of interleukin 4 receptor (IL-4R) on methylcholanthrene (MCA-106, MCA-102, and MC-38)- and viral DNA (G-2TS and 14-2TS)-induced murine sarcoma cells was demonstrated. MCA-106 tumor cells express about 500 to 1348 (median, 800) interleukin 4 (IL-4) binding sites/cell with a dissociation constant (Kd) of 115 +/- 26 pM (mean +/- SD, n = 4). By Northern blot analysis, tumor cells exhibited a single mRNA species of 3.9 kilobases. Other murine sarcoma (MCA-102), colon adenocarcinoma (MC-38), G-2TS, and 14-2TS tumor cells express low numbers of IL-4R. By immunoperoxidase staining, 81 to 92% of the cells from fresh MCA-106 tumors were positive for IL-4 receptors, while only 7 to 10% of tumor-infiltrating cells were Thy 1.2 and less than 1% Mac-1 positive. Using a chimeric protein composed of IL-4 and Pseudomonas exotoxin (IL-4-PE40), we observed that IL-4-PE40 was cytotoxic (determined by inhibition of protein synthesis by [3H]leucine uptake) to MCA-106 tumor cells in a dose-dependent manner. A nonchimeric protein (PE40) that cannot bind to the IL-4R did not inhibit protein synthesis in tumor cells. A chimeric mutant protein (IL4-PE40 asp553) that can bind to IL-4 receptors but does not have the capability to inhibit protein synthesis was not cytotoxic to tumor cells. These studies strongly suggest that IL-4R on murine MCA-106 sarcoma cells is internalized when occupied by IL-4 PE40. Furthermore, a neutralizing antibody (11B11) to IL-4 completely abolished the protein synthesis-inhibitory activity of IL-4-PE40. G-2TS tumor cells which expressed low numbers of IL-4 receptors were not vulnerable to cytotoxicity by IL-4-PE40. Taken together, these data suggest that IL-4 receptor may be a target for IL-4-toxin therapy.
Water supplementation in exclusively breastfed infants during summer in the tropics.
This study was designed to determine the need for water supplementation to maintain water homoeostasis in exclusively breastfed infants during summer in a tropical country. A prestudy questionnaire revealed that 97% of 34 nurses and 63% of 70 doctors advocated such supplementation. 45 healthy, male, exclusively breastfed babies, aged 1-4 months, were recruited from a well-baby clinic. 9 who had never received supplemental water plus a random selection of 14 others were allocated to group I (breastmilk only); the remaining 22 infants were allocated to group II (breastmilk plus supplemental fluid according to the mother's usual practice). The babies were studied at the hospital for 8 h; breastmilk intake was measured by weighing the infant before and after each feed, water intake by calibrated bottles, and urine output by accurate collection and measurement. The maximum room temperatures were 34-41 degrees C and relative humidities 9-60% (below 50% in all but 3 infants). In group II the mean water intake was 11% (95% confidence interval 7-16%) of the total fluid intake. Both breastmilk intake (274 vs 210 ml) and total fluid intake (274 vs 233 ml) were higher in group I than in group II (p = 0.003, p = 0.073, respectively), after adjustment for age, weight, length, room temperature, and humidity. However, there were no significant differences between the groups in urine output, urine or serum osmolality, weight change, or rectal temperature whether or not the factors adjusted for included total fluid intake. Thus, exclusively breastfed infants do not need supplemental water to maintain water homoeostasis; a reduced breastmilk intake is a potential disadvantage of this practice.
Reversible computerized tomographic lesion following childhood seizures.
Recently reversible computerized tomographic (CT) lesions following seizures have been reported, mostly in adult subjects. Focal CT lesions following seizures were recorded in 20 children. The distribution of the lesions was parietal in 17, frontal in two, occipital in one, and temporo-parietal in one. Multiple simultaneous lesions were observed in one child. Plain scan revealed evidence of cerebral oedema in 16 patients (mild 10, moderate four, and severe two). Enhanced scan showed a ring lesion in 12, hyperdense focus in five, and non-enhancing (hypodense) lesion in two children (excluding one child who had multiple lesions; three rings and one dense focus). These subjects were treated with anti-epileptic drugs only. Repeat CT scans yielded a complete resolution in 12 and a significant resolution in eight. It is concluded that in children with seizures, reversible CT demonstrable focal abnormalities may occur.
Does breastfeeding influence mortality in children hospitalized with diarrhoea?
The association between breastfeeding and mortality in children hospitalized for diarrhoea was investigated in a prospective manner in 309 subjects below 18 months of age. In multivariate logistic regression analysis, 36 cases who died were compared with 273 controls who were discharged in a satisfactory condition. Breastfeeding had a strong protective effect against mortality even after allowance was made for confounding variables (including nutritional status, chronicity of illness, associated non-enteral infections) and a possible bias of interruption of breastfeeding as an early consequence of the terminal illness. The adjusted odd's ratio (OR) and 95 per cent confidence intervals (95 per cent CI) for the protective effect were 2.7 and 2.1-3.6, respectively. The adjusted OR's (95 per cent CI's) were 6.0 (3.6-10.2), 2.6 (2.0-3.4), and 1.8 (1.4-2.5) for the age intervals 0-6, 7-12, and 13-18 months, respectively (P less than 0.001, less than 0.01, and less than 0.05, respectively). Further stratified analyses suggested a greater benefit in children with severe wasting, severe stunting, protracted illness, and diarrhoea as the sole illness. It is concluded that in children up to 18 months of age, breastfeeding offers substantial protection against death in children hospitalized with diarrhoea.
In vivo proliferation of adoptively transferred tumor-infiltrating lymphocytes in mice.
The adoptive transfer of tumor-infiltrating lymphocytes (TILs) in conjunction with interleukin-2 (IL-2) administration can mediate a reduction in established pulmonary and hepatic metastases of a variety of murine tumors as well as in patients with metastatic melanoma. To characterize further the fate of adoptively transferred TILs, the uptake of the thymidine analog 5-[125I]iodo-2-deoxyuridine ([125I]UdR) into the DNA of dividing cells was used to study the in vivo proliferation and migration patterns of transferred TILs in C57BL/6N mice. Animals received 500 rad of total body irradiation prior to cell transfer to separate incorporation of radiolabel into endogenous lymphoid cells from that into transferred TILs. Mice were subsequently treated with i.v. injections of TILs or no cells followed by i.p. injections of Hanks' balanced salt solution or IL-2. At various time points, mice received [125I]UdR, and 20 h later tissues were removed and counted on a gamma analyzer. A proliferation index (PI) was calculated by dividing the mean cpm of organs of experimentally treated mice by the mean cpm of organs of control mice. Animals receiving TILs alone demonstrated small increases in [125I]UdR in the lungs, liver, and spleen of saline-treated controls (PI = 1.4, 1.6, and 1.7, respectively, on day 4), while animals treated with 50,000 U of IL-2 alone showed greater increases in the lungs, liver, kidneys, and spleen (PI = 3.9, 6.1, 3.3, and 15.8). Mice receiving TILs plus IL-2 demonstrated the highest levels of radiolabel incorporation in the same organs (PI = 10.5, 19.4, 10.2, and 22.4). Over a period of 10 days, TIL plus IL-2 treated animals continued to incorporate significantly greater amounts of [125I]UdR for as long as high-dose IL-2 was administered. Animals treated with TILs demonstrated increased incorporation of radiolabel with increasing doses of IL-2. Injection of irradiated TILs did not result in an increased uptake of [125I]UdR into these tissues, thus confirming that TIL proliferation is responsible for the radiolabel uptake in animals receiving TILs alone or TILs plus IL-2. Additionally, fluorescein-labeled anti-Thy-1.1 antibody identified proliferating TILs derived from congenic B6.PL Thy 1a/CY (Thy-1.1) animals in the lungs, spleen, and liver of recipient C57BL/6N (Thy 1.2) mice. In summary, we have demonstrated that adoptively transferred TILs distribute widely after i.v. injection and can proliferate in various tissues especially under the influence of exogenous IL-2.
Risk factors for fatal diarrhea in hospitalized children in India.
There is scant information on the risk factors for diarrheal deaths in developing countries. A prospective evaluation was therefore conducted on 382 consecutive children (less than 5 years of age), who were hospitalized with diarrhea. A complete profile, including outcome, was available for 357 patients, 37 of whom died (10.4%). In the univariate analysis, four factors were significantly associated (p less than 0.02) with death: associated major infection (pneumonia, septicemia, or meningitis), severe wasting (less than or equal to 50% weight for age), severe stunting (less than or equal to 85% height for age), and protraction of illness (greater than 14 days). In the multivariate analysis, all four factors retained their significance. The adjusted odds ratios (95% confidence interval) were 4.7 (3.9, 5.6), 3.3 (2.7, 4.0), 1.9 (1.6, 2.3), and 1.5 (1.3, 1.8), respectively. In addition, in children less than 19 months of age (n = 241; 29 deaths) breast-feeding had a significant (p less than 0.001) protective effect (adjusted OR--2.3, 95% CI-1.9-2.8). It is concluded that even in a setting with high diarrheal fatality rates, high-risk children can be identified and targeted for intensive intervention.
Antigen(s)-specific tumour-infiltrating lymphocytes from tumour induced by human herpes virus-6 (HHV-6) DNA transfected NIH 3T3 transformants.
Tumour infiltrating lymphocytes (TIL) have recently been shown to mediate potent therapeutic effects in certain malignancies in mice and in humans. To understand the mechanism of TIL immunotherapy it would be advantageous to generate tumour-specific TIL and to study a defined system of TIL and target cells in which the tumour epitope(s) recognized by TIL might be identified. We have established tumourigenic cell lines by transfection of NIH 3T3 cells with the entire genome of human herpesvirus-6 (HHV-6) and its small fragment (about 5% of the viral DNA sequence). Injection of these cells into nude mice produced tumours termed G-2T and 14-2T, respectively. Cell lines derived from these tumours when injected in NIH Swiss mice produced tumours, G-2TS and 14-2TS, respectively. We have generated TIL from G-2TS tumour that can kill G-2TS tumour cells in vitro but not other related tumours (14-2TS or MCA-106). These TIL can be expanded between 2-6.5 every 3-5 days. The TIL proliferated in tissue culture in response to recombinant interleukin-2 and interleukin-4 and maintained their tumor specificity for up to 6 months in vitro. Their phenotype was Thy 1.2+, Lyt-2+ and L3T4-. The availability of such tumour-specific stable TIL lines and specific viral-transformed targets will provide an opportunity to characterize the tumour-associated antigen critical for the specific cytotoxicity in this system and thereby to clarify the mechanism of this promising immunological approach to cancer therapy.
In vivo treatment with interferon causes augmentation of IL-2 induced lymphokine-activated killer cells in the organs of mice.
Interferon-alpha (IFN-alpha) has been shown to synergize with IL-2 in the regression of a variety of established murine tumours and studies are underway to explore this combination in patients with advanced cancers as well. To understand the mechanism of synergy we have studied lymphokine-activated killer (LAK) cell activity in various compartments of mice in response to IFN-alpha and IL-2 administration. The effects of IFN-gamma, TNF-alpha and IL-4 were also examined. C57BL/6 mice were injected intraperitoneally with HBSS, IL-2 alone, IFN-alpha alone or both, two times a day for 7 days. On days 4 and 8, LAK activity was tested in a 4-h chromium release in cells obtained from lungs, spleen, and liver using fresh MCA-102 tumour cells as targets. The cells from control mice failed to lyse the MCA-102 target. IL-2 caused the generation of LAK activity and an increase in total cell yield in all the organs after 3 days of injection. IFN-alpha failed to generate LAK activity but when administered along with IL-2, caused synergistic enhancement of LAK lysis of MCA-102 target cells. Cell yield in this group was lower as compared with the IL-2-treated group. LAK activity tested after 7 days of IL-2 therapy was significantly decreased compared with that observed after 3 days. However, activity remained at as high a level after 7 days of therapy as after 3 days of therapy in animals treated with IFN-alpha and IL-2. FACS analysis revealed that asialo GM-1+ (ASGM-1) and NK1.1+ cells were increased in number in IL-2 and IL-2 plus IFN-alpha-treated spleen; however, the number of these cells was similar in both groups. In the liver, ASGM-1+ cells were higher in the IL-2 plus IFN-alpha group than in the group treated with IL-2 alone. By in vitro depletion utilizing antibody and Rbc' experiments, it was clear that both ASGM-1+ and NK1.1+ cells from the spleen mediated most of the cytotoxicity of MCA-102 targets. Pre-treatment irradiation (5 Gy) of mice completely abrogated the capability of IL-2 or IL-2 plus IFN-alpha to generate LAK activity. IFN-gamma also had a stimulatory effect on IL-2 induction of LAK activity. Tumour necrosis factor-alpha (TNF-alpha) and IL-4 failed to generate LAK activity and, in combination with IL-2, no additional stimulatory effect was observed.(ABSTRACT TRUNCATED AT 400 WORDS)
Interleukin-2 toxicity.
Because of the ability of interleukin-2 (IL-2) to support the proliferation and activation of numerous types of immunocompetent cells and to support the survival of adoptively transferred lymphocytes, there has been considerable interest in its use in the immunotherapy of malignancies. While studies to date have indicated that IL-2 has activity against some malignancies, considerable toxicity has also been observed. Careful prescreening and selection of patients and appropriate management of toxicity can minimize adverse outcomes. Studies of IL-2 effects have provided intriguing evidence of interactions of the immune/cytokine system with the neuroendocrine, cardiovascular, and other systems. Studies in animal models have demonstrated the central role of an intact immune system in mediating many toxicities of IL-2. Several adverse effects of IL-2 appear to be mediated by other cytokines whose production is induced by IL-2. Studies into the pathogenesis and manifestations of IL-2 toxicity have offered the hope of developing less toxic approaches to IL-2 therapy. Several lessons from the IL-2 experience are likely to be applicable in the clinical development of other cytokines.
Antilymphocyte and antithymocyte globulin.
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A profile of pyogenic meningitis in children.
Clinical profile of pyogenic meningitis was studied in 256 cases over a period of 8 years. The male to female ratio was 1.46: 1 and 83.6% of the patients were less than 3 years of age. Fever, altered sensorium, refusal to feed, convulsions and vomiting were the common presenting symptoms. Six (2.3%) of them had subnormal temperature and the sensorium was normal in 12.5% of cases. Cerebrospinal fluid was clear in 9.4%, cell count was less than 100/cmm in 8.2%, sugar was more than 40 mg% in 24.2% and protein was less than 50 mg% in 12.5% of cases. Diplococcus pneumoniae was the commonest causative agent. The overall mortality was 30.5%. It is concluded that young children suffering from this disease may not have the typical features and one should have the highest suspicion to make an early diagnosis so that the morbidity and mortality from this condition can be reduced.
Charter for care of children in hospitals.
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Mortality in neonatal septicemia with involvement of mother in management.
In most Special Care Neonatal Units (SCNUs) in India, mothers are excluded from the care of their sick babies for fear of over-crowding and dislocation. We have attempted to study the feasibility of involving mothers in the care of their babies admitted for neonatal septicemia and to analyse whether this changed the sepsis related case fatality rate. The study material consisted of 158 neonates with blood culture positive neonatal septicemia whose mothers were actively involved in their care during their stay in the SCNU of LNJPN Hospital throughout 1987-88. The mothers lived in with their sick neonates and were extremely useful in feeding, cleaning, and monitoring for some important signs and symptoms. There were no epidemics of infection in the nursery during this period. All the babies discharged were receiving breast feeds, and the mothers were confident in taking care of them before discharge. The mortality in this group was 43%. The onset of septicemia was most often in the first week (36%) being 25.9% in second week, 26.6% in the third, and 11.4% in the fourth. Mortality was maximum (64.5%) when the onset of illness was in the first 3 days. Klebsiella and S. aureus were commonly isolated organisms (38.6 and 21.5%, respectively). Gram negative organisms were isolated in 66.5% cases with higher mortality in this group. Nearly 46% of the babies weighed 2 kg or less, with a mortality of 60.2% compared to 28.2% in those more than 2 kg. Only 3 to 5% and 40 to 66.7% of Gram negative and 23 and 70% of Gram positive organisms were sensitive to ampicillin and gentamicin, respectively.
Neonatal shigellosis.
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Women's health and fetal outcome.
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