Biomedical subjects
Publications and source records attributed to G Krishnaswamy.
A patient presented with severe bactrim-induced neutropenia with a reversed CD4+/CD8+ lymphocyte ratio. R-metHUG-CSF at 300 micrograms daily produced a dramatic neutrophil response and the therapy was discontinued after 2 weeks.
The expression of prostaglandin H synthases can be induced by many stimuli and is likely to be important in control of the cell cycle. The analysis of prostaglandin H synthase-1 and -2 expression in colon adenocarcinoma cell lines is a useful model system for studying the function of the prostaglandin H synthases, especially with regard to proliferation and adhesion. Prostaglandin H synthase-1 protein is not found in any of eight human colon adenocarcinoma cell lines. Expression of prostaglandin H synthase-2 is variable for the eight cell lines: three constitutively expressed active protein, four did not express this gene at all, and one had mRNA but no active protein. Thus, five colorectal adenocarcinoma cell lines exhibit "null" expression of prostaglandin synthase-2. The three cell lines with constitutive expression of prostaglandin H synthase-2 produce PGE2. Prostaglandin E2 production could be inhibited by aspirin and NS398 without inhibiting proliferation, while direct addition of prostaglandin E2 inhibits proliferation. Adhesion to collagen IV and fibronectin was stronger in those cell lines that expressed prostaglandin H synthase-2. The constitutive expression of prostaglandin H synthase-2 is associated with increased adhesion to extracellular matrix components and a potential inhibition of proliferation through the production of prostaglandin E2. The absence of PGH synthase-2 expression in some cell lines may result from the original tumor's need to inactivate these associated functions. Our evidence suggests that PGH synthase-2 is a possible candidate for a tumor suppressor gene at 1q23-qter.
The pathophysiology of allergic disease is multifactorial, involving an intricate network of interactions among cells, mediators, and cytokines. Substantial progress has been made in defining the role of antigen-specific T cells and cytokines in the regulation of immunoglobulin E (IgE) synthesis and the atopic diseases. The development of antigen-specific T-cell lines and clones has facilitated efforts to characterize human T-cell subsets and their cytokine repertoires. Molecular methods currently available include techniques for the quantitative analysis of cytokine gene expression and secretion from activated T cells ex vivo as well as in tissues. The availability of these newly developed techniques has become essential to the investigation of the pharmacologic regulation of T cells and cytokines both in vitro and in vivo. Future investigations will contribute to our understanding of the differential regulation of T-cell subsets and their relationships to allergic diseases, ultimately leading to a better understanding of the molecular pathogenesis of allergic diseases and the design of more effective therapeutic interventions.
Human mast cells readily release a variety of mediators, including cytokines, in response to IgE receptor crosslinking, but the mechanisms governing the expression of cytokines are still unclear. Using a human mast cell line, HMC-1, we show expression of cytokine transcripts as early as 2 h after activation with ionomycin and phorbol myristate acetate (PMA). Resting HMC-1 cells expressed transcripts for interleukin-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-5, GM-CSF, and weakly for IL-8, and stimulation with ionomycin and PMA induced additional transcripts for IL-6 and IL-13 and upregulated expression of IL-8 transcripts. HMC-1 cells secreted IL-4, IL-8, and GM-CSF protein after activation and dexamethasone significantly inhibited the production of these cytokines. Of significance is the finding that the addition of membranes purified from activated T cells to mast cell cultures induced transcripts selectively for IL-8 and none for other proinflammatory cytokines. Flow cytometry revealed that resting HMC-1 cells express CD40, a molecule involved in contact-dependent signaling of monocytes and B cells by T cells. However, activation of HMC-1 by anti-CD40 antibody did not induce IL-8 gene expression or protein production. This study demonstrates that human mast cells are capable of expressing multiple cytokines that can be inhibited by glucocorticoids. It also raises the possibility that T cells may activate mast cell cytokine synthesis by novel contact-dependent mechanisms. This phenomenon of T cell regulation of mast cell function requires further study.
We have recently developed a new PCR assay for the detection of H. pylori. In this study, the polymerase chain reaction (PCR) assay was used to detect H. pylori in 88 gastric biopsy, 85 saliva, and 71 fecal specimens from 88 patients. H. pylori infection was confirmed in 71 of 88 patients by culture and/or histological stain of gastric biopsies. Serum IgG antibody to H. pylori was also measured and resulted in 97% sensitivity and 94% specificity. H. pylori DNA was detected by the PCR assay in gastric biopsy specimens from all 71 patients (100% sensitivity) with proven gastric H. pylori infection but not from 17 noninfected patients (100% specificity). In saliva specimens, H. pylori DNA was identified in 57 of the 68 patients (84%) with proven gastric H. pylori infection and in three of the 17 patients without gastric H. pylori infection. However, the PCR assay was only able to detect H. pylori DNA in the feces from 15 of 61 patients (25%) with proven gastric H. pylori infection and one of the 10 patients without gastric H. pylori infection. The results show that the PCR assay is reliable for detecting the presence of H. pylori in gastric biopsy and saliva specimens. The data indicate that H. pylori exists in a higher prevalence in saliva than feces and that the fecal-oral route may be an important means of transmission of this infection in developing countries but not as significant as previously suspected in the developed countries. It is likely that the oral-oral route is more prominent.
This case demonstrates the importance of recognizing urticarial vasculitis in patients with chronic urticarial eruptions. The salient points in history that point towards the diagnosis of urticarial vasculitis include the presence of painful urticarial lesions that last longer than 24 hours and that heal leaving residual pigmentation. In some cases the urticaria may evolve into palpable purpura. An associated systemic illness that may resemble systemic lupus erythematosus should also suggest the diagnosis, which is established by skin biopsy. Histopathology reveals a leukocytoclastic vasculitis involving postcapillary venules. When associated with systemic vasculitis, the urticaria is likely to be of the hypocomplementemic variety, with immunoglobulin and complement deposition on biopsies, and with serum complement studies demonstrating classical pathway activation, low C1q levels, and anti-C1q precipitins. A variety of agents have been used in the management of urticarial vasculitis, including aspirin, nonsteroidal anti-inflammatory agents, corticosteroids, colchicine, dapsone, hydroxychloroquine, and cytotoxic agents such as cyclophosphamide and azathioprine.
T cell-derived cytokines, such as interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) activate eosinophils, whereas other cytokines, such as tumor necrosis factor (TNF)-alpha and IL-13, determine eosinophil recruitment. Interferon-alpha (IFN-alpha), a leukocyte-derived cytokine, has been shown to have beneficial effects in eosinophil-mediated disorders, such as the hypereosinophilic syndrome and a murine model of allergic asthma, where it inhibited eosinophil recruitment. We tested the hypothesis that IFN-alpha acted in eosinophil-mediated disorders by modulating T cell cytokine expression. Peripheral blood mononuclear cells (PBMC) or human ragweed-specific TH1 (2B8) and TH2 (2D2) T cell clones were cultured in the presence of 5 micrograms/ml of phytohemagglutinin (PHA) or 25 micrograms/ml of antigen Amb a 1 (short ragweed allergen), respectively, and lymphoblastoid IFN-alpha (varying from 0 to 10,000 U/ml). We assessed T cell proliferation by [3H]thymidine incorporation and production of IL-5 and GM-CSF by ELISA. Expression of cytokine transcripts was analyzed by the reverse transcription-polymerase chain reaction technique (RT-PCR). IFN-alpha induced a dose-dependent suppression of T cell proliferation of both PBMC (p < 0.001) and the T cell clones (p < 0.001). IFN-alpha inhibited gene expression of IL-5, GM-CSF, TNF-alpha, and IL-13 in PBMC. Furthermore, IFN-alpha significantly inhibited mitogen-induced and antigen-induced production of IL-5 and GM-CSF. IFN-alpha may benefit eosinophil-mediated disorders by inhibiting T cell function and production of cytokines active on human eosinophils.
Felty's syndrome is characterized by neutropenia, splenomegaly, and recurrent infection in patients with rheumatoid arthritis. We used recombinant granulocyte colony stimulating factor (rGCSF) in a patient with Felty's syndrome and recurrent sepsis. rGCSF induced a statistically significant increase in the patient's absolute neutrophil and total white blood cell counts. During 14 months of followup taking rGCSF, disseminated varicella zoster was the only infectious complication. Except mild thrombocytopenia and a transient flare of arthritis, no serious adverse effects occurred. rGCSF may be a safe and effective therapy for Felty's syndrome in selected patients.
/We have studied the effect of interferon alpha (IFN-alpha) on MHC class II expression by human buccal epithelial cells (BEC), and mRNA expression by BEC and mucosal-associated mononuclear cells (MAMC). In 6 experiments, freshly collected BEC were suspended at a concentration of 1.0 x 10(5)/ml in RPMI 1640 and incubated in the presence of 0-10,000 IU/ml of human lymphoblastoid IFN-alpha (HuIFN-alpha). Zero and six hour samples were analyzed by single color flow cytometry using FITC-labeled murine IgG1 monoclonal antibody to HLA-DR. Preparations were also analyzed for expression of cytokine transcripts (IL-2, IL-4, IL-5, IL-6, IL-8, IFN-gamma, GM-CSF) by reverse transcriptase polymerase chain reaction (RT-PCR). Increasing concentrations of IFN-alpha resulted in proportionate increases in the percentage of HLA-DR + BEC (r = 0.7897, p = 0.0627) and in the percentage of HLA-DR+ staining at higher intensities (10(1) to 10(2) log fluorescence intensity) (LFI) (r = 0.4010, p = 0.0424). The percentage of HLA-DR + BEC rose from a mean of 1.5% with no IFN-alpha to 7% with 10,000 IU/ml IFN-alpha (p < 0.05). The percentage of HLA-DR + BEC staining at 10(1) to 10(2) LFI rose from a mean of 8.3% with no added IFN-alpha to 19.2% with 10,000 IU/ml IFN-alpha (p < 0.05). Unstimulated BEC constitutively expressed IL-8 and GM-CSF. IFN-alpha stimulated preparations also expressed IFN-gamma, possibly due to the presence of MAMC, which comprised 2-9% of the total cell population. These data indicated that HuIFN-alpha upregulates MHC class II expression by human BEC, possibly by enhancing IFN-gamma production by MAMC present in the culture preparations.
Drug-resistant tuberculosis (DRTB) is a growing national health concern in both urban populations and rural areas and is exacerbated by the growing epidemic of human immunodeficiency virus (HIV) infection. Between 1989 and 1992, 7 cases of DRTB (5 with multidrug-resistance) were diagnosed in an eight-county region of East Tennessee. During 1990 and 1991 alone, 5 of 100 patients with tuberculosis had drug-resistant strains (5%). All 7 patients with DRTB had 100% resistance to isoniazid; 5 also had resistance to streptomycin, 2 to rifampin, and 1 to pyrazinamide and ethambutol. All patients were white, U.S.-born, and without evidence of HIV infection. Contact investigation revealed that more contacts of patients with DRTB (13 of 74, 18%) were infected than were contacts of patients with drug-sensitive tuberculosis (46 of 290, 16%). Our study demonstrates that DRTB is not confined to geographically distinct areas, but may be a subtle and easily missed diagnosis in presumably low-risk rural populations.
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Sib-pair analysis of 170 individuals from 11 Amish families revealed evidence for linkage of five markers in chromosome 5q31.1 with a gene controlling total serum immunoglobulin E (IgE) concentration. No linkage was found between these markers and specific IgE antibody concentrations. Analysis of total IgE within a subset of 128 IgE antibody-negative sib pairs confirmed evidence for linkage to 5q31.1, especially to the interleukin-4 gene (IL4). A combination of segregation and maximum likelihood analyses provided further evidence for this linkage. These analyses suggest that IL4 or a nearby gene in 5q31.1 regulates IgE production in a nonantigen-specific (noncognate) fashion.
Human cytokines, IL-4, IL-5, and IFN-gamma play an important role in the regulation of IgE synthesis and atopic diseases. In this communication, we describe the development of a quantitative assay of steady-state cytokine mRNAs (IL-4, IL-5, and IFN-gamma) from a variety of cell sources, including peripheral blood mononuclear cells (PBMCs) stimulated with either a mitogen (PHA) or ragweed pollen allergen extract, and cells from allergen-challenged inflammatory sites. Quantitative analysis of IL-5, IL-4 and IFN-gamma transcripts was achieved by a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique using internal standard (IS) cRNAs in the presence of specific oligonucleotide primers. Each IS was generated from a plasmid vector containing the respective cytokine cDNA modified by insertion with an SV40-DNA fragment. Both test RNA and IS were reverse-transcribed and subjected to the 'competitive' PCR in the same tube. We first demonstrate the linearity and reproducibility of this technique; second, we apply this competitive PCR assay to analyze quantitatively the expression of IL-4, IL-5, and IFN-gamma transcripts in PBMCs before and after stimulation with PHA or crude ragweed allergen. Finally, we analyzed cells isolated from the lung lavage fluids of an atopic subject following allergen challenge, and showed a significant increase of IL-4 and IL-5 transcripts, but not IFN-gamma, in the allergen-challenged site when compared to the control. This technique of PCR quantitation provides an easy and efficient tool to study the expression of cytokine genes in allergic inflammatory diseases.
Variation in sensitivity to cisplatin during the cell cycle was studied in synchronous Chinese hamster ovary cells treated during G1 or late S for 1 h at 41.5 degrees C with cisplatin (0.25-1.25 micrograms/ml, 0.8-4.2 x 10(-6) M). The cells were assayed for cell killing and chromosomal aberrations. Either they were plated for colony survival, or colcemid was added from 12 to 40 h after plating followed by fixation 4 h later for analysis of chromosomal aberrations after the cells completed 1 or 2 cycles (i.e., first or second mitosis). When the cells were treated either in G1 or late S, the cells entering metaphase exhibited primarily chromatid-type deletions and exchanges. However, aberrations were observed primarily in the first mitosis when cells were treated in G1 compared with aberrations being observed in both the first and second mitoses when cells were treated in late S. For a given amount of cytotoxicity or cytological damage, the cisplatin concentration at 41.5 degrees C could be reduced 4-6-fold compared with treatment at 37 degrees C. For low cisplatin concentrations of less than 0.5-0.7 microgram/ml (survival, approximately 0.3), heat killing predominated, and cells treated in S phase were more sensitive than those treated in G1. However, for cisplatin concentrations greater than 0.5-0.7 microgram/ml, cisplatin cytotoxicity predominated, and for both cell killing and chromosomal aberrations, the cells treated in G1 were approximately 1.5 times more sensitive than those treated in late S. Furthermore, the positive correlation between survival and aberration frequency was similar for cells treated at 37 degrees C or 41.5 degrees C in either G1 or late S. These results suggest that cisplatin administered at 37 degrees C or 41.5 degrees C causes cell lethality primarily by the induction of chromosomal aberrations.
Variation in sensitivity to cisplatin during the cell cycle was studied in synchronous CHO cells treated during G1 or late S phase. The cells were assayed for cell killing, cell-cycle delay, and chromosomal aberrations after they were treated with cisplatin (1-12 micrograms/ml) for 1 h at 37 degrees C. They were either plated for colony survival, or colcemid was added from 12 to 40 h after plating followed by fixation 4 h later for analysis of chromosomal aberrations after the cells completed 1 or 2 cycles (i.e. first or second mitosis). Cells treated with 6 micrograms/ml exhibited about a 10-h delay during the first cycle after treatment during G1 compared with about 3 h during the first cycle and 6 h during the second cycle after treatment during late S. In both cases, cells entering metaphase exhibited predominantly chromatid-type breaks and exchanges. For both cell killing and chromosomal aberrations, the cells in G1 were 1.5-1.6 times more sensitive than those treated in late S, with 1 aberration per cell corresponding to about 37% survival. However, the exchanges and breaks were observed primarily in the first mitosis when cells were treated in G1 compared with the second mitosis when cells were treated in late S. These results suggest that DNA replication opposite cisplatin cross-links in the DNA results in lethal chromosomal aberrations.
The purpose of this study was to determine whether there are differences in bone mineral content and density between paralyzed and nonparalyzed sides of patients who had sustained strokes associated with unilateral muscle weakness, to determine the relationship between duration of stroke and degree of demineralization and to compare the degree of demineralization in upper and lower limbs. The bone mineral content and density were measured by dual photon absorptiometry (high resolution scanning mode, Lunar DP4) in ambulant patients with a history of single completed strokes associated with unilateral weakness. The bone mineral content and density of each limb was determined by the region of interest analysis program. In the 30 patients included in this study, the bone mineral content and density were significantly less on the paralyzed than on the nonparalyzed side. The degree of demineralization was more pronounced in the upper than in the lower limbs. The mean percentage differences in bone mineral content and density between paralyzed and nonparalyzed arms were 13.8% (P < 0.00001) and 7.95% (P = 0.0003), respectively, and between paralyzed and nonparalyzed legs the differences were 4.5% (P = 0.0012) and 3.42% (P = 0.0028), respectively. A better correlation was noted between the time elapsed since the stroke and the degree of demineralization in the upper limbs (r = 0.75, P < 0.0001), than in the lower limbs (r = 0.60, P = 0.0004). In conclusion, patients who have strokes associated with muscle weakness are at an increased risk of developing osteoporosis on the paralyzed side and particularly in the upper limbs.
A panel of steady-state cytokine mRNAs was analyzed in the bronchoalveolar lavage (BAL) cells from asthmatic subjects or patients challenged with ragweed allergen. This was achieved by combining both qualitative and quantitative assays using the reverse transcription-polymerase chain reaction (RT-PCR). Analysis of BAL cells from six mild allergic asthmatic and five nonasthmatic, nonallergic subjects showed no qualitative differences in the profile of cytokine mRNAs (including interleukin [IL]-1 beta, IL-2, IL-5, IL-6, IL-8, and granulocyte/macrophage colony-stimulating factor), except for tumor necrosis factor-alpha, which was detected in three out of six asthmatic BAL samples but in none of the controls. A key cytokine, IL-5, has been implicated in the pathogenesis of allergic inflammation through the recruitment of eosinophils. We found a significant enhancement of steady-state IL-5 transcripts in the BAL cells from allergen-challenged as compared with the saline-challenged control sites of four asthmatic patients; furthermore, the cellular source for IL-5 mRNA was identified in the mononuclear cell fraction, but not in the purified eosinophils, of the allergen-challenged BALs. These results suggest that the significant increase of IL-5 transcripts is primarily from the infiltrating mononuclear cells. Our study also demonstrates the power of qualitative and quantitative PCR analysis in determining the molecular basis of allergic inflammatory diseases.
The following four effects on DNA replication are observed in cells heated in S phase of the cell cycle: (1) inhibition of replicon initiation, (2) delay in DNA chain elongation into multicluster-sized molecules > 160S, (3) reduction in fork displacement rate, and (4) increase in single-stranded regions in replicating DNA. Since cells heated in S phase manifest chromosomal aberrations when they enter metaphase, whereas cells heated in G1 do not, we attempted to determine if the effects on DNA replication are critical for the induction of chromosomal aberrations by studying these same effects during DNA replication when synchronous CHO cells had been heated (10 min at 45.5 degrees C) in G1 phase. Following a heat-induced G1 block (12 h), we found previously that when the cells entered S phase, replicon initiation was functional and chain elongation into multicluster-sized molecules > 160S was delayed but completed during S phase. In the present study, we find that the fork displacement rate was near normal and that there was no increase in single-stranded DNA. Additionally, an increase in excess nuclear protein induced in the heated G1-phase cells returns to a normal level by about 12 h, just prior to when the cells enter S phase. Since the excess nuclear protein remains for many hours in heated S-phase cells, we hypothesize that the excess nuclear protein is responsible for the drastic reduction in the fork displacement rate and the associated increase in single-stranded DNA. Furthermore, we hypothesize that this persistent increase in single-stranded DNA during replication is a critical step for the induction of chromosomal aberrations in heated S-phase cells. Consistent with this hypothesis, we observed that aphidicolin (1-2 micrograms/ml) treatment of S-phase cells for 13-16 h, which results in a twofold increase in single-stranded DNA during the inhibition of DNA synthesis, also induces chromosomal aberrations. Possibly, endogenous endonucleolytic attack occurs opposite these sites of single-stranded DNA, thus creating double-strand breaks which either can remain unrepaired or are misrepaired to account for the chromatid breaks and exchanges, respectively, observed as cells complete their cell cycle and enter metaphase.