Geographic information system-based analysis of COVID-19 cases in India during pre-lockdown, lockdown, and unlock phases.

OBJECTIVE: The World Health Organization formally announced the global COVID-19 pandemic on March 11, 2020 due to widespread infections. In this study, COVID-19 cases in India were critically analyzed during the pre-lockdown (PLD), lockdown (LD), and unlock (UL) phases. METHOD: Analyses were conducted using geospatial technology at district, state, and country levels, and comparisons were also made with other countries throughout the world that had the highest infection rates. India had the third highest infection rate in the world after the USA and Brazil during UL2.0-UL3.0 phases, the second highest after the USA during UL4.0-UL5.0 phases, and the highest among South Asian Association for Regional Cooperation (SAARC) countries in PLD-UL5.0 period. RESULTS: The trend in the number of COVID-19 cases was associated with the population density where higher numbers tended to be record in the eastern, southern, and west-central parts of India. The death rate in India throughout the pandemic period under study was lower than the global average. Kerala reported the maximum number of infections during PLD whereas Maharashtra had the highest numbers during all LD and UL phases. Eighty percent of the cases in India were concentrated mainly in highly populous districts. CONCLUSION: The top 25 districts accounted for 70.99%, 69.38%, 54.87%, 44.23%, 40.48%, and 38.96% of the infections from the start of UL1.0 until the end of UL phases, respectively, and the top 26-50 districts accounted for 6.38%, 6.76%, 11.23%, 12.98%, 13.40%, and 13.61% of cases in these phase, thereby indicating that COVID-19 cases spread during the UL period. By October 31, 2020, Delhi had the highest number of infections, followed by Bengaluru Urban, Pune, Mumbai, Thane, and Chennai. No decline in the infection rate occurred, even in UL5.0, thereby indicating a highly alarming situation in India.

Development of a Taqman-based real-time PCR assay for detection of porcine sapelovirus infection in pigs.

The objective of the present study was to develop a rapid, simple, specific and sensitive Taqman-based real-time PCR assay for porcine sapelovirus (PSV) detection. Specific primers and probe were designed from the five untranslated regions (UTRs) of the viral genome. The detection limit of the real-time PCR was 102 copies. The specificity of the Taqman real-time PCR assay was evaluated using other animal viruses and nuclease free water as a negative control. Strong fluorescent signals were obtained only in the detection of PSV real-time PCR and conventional RT-PCR were preformed simultaneously on 90 faecal samples. Based on conventional RT-PCR study 17.7% (16/90) of the faecal samples were positive for PSV. Whereas 21 of 90 samples (23.3%) were positive by real-time RT-PCR. The results showed that real-time PCR was more sensitive than the conventional RT-PCR assay. In conclusion, the Taqman real-time PCR assay for detection of PSV developed, herein, is sensitive, specific, and reliable. This assay will be useful for clinical diagnosis, epidemiological, and pathogenesis studies.

Immunocytochemistry assay in BHK-21 cell line infected with Porcine Sapelovirus.

The present study describes an immunocytochemistry (ICC) assay with self-raised hyperimmune sera and a Baby Hamster Kidney-21 (BHK-21) cell line infected with Porcine Sapelovirus (PSV). Sapelovivus/IVRI/SPF-c-6/2015 strain Indian PSV was isolated from the porcine IBRS-2 cell line and investigated for growth on non-porcine cell lines. After two passages, PSV was successfully grown in BHK-21 and produced the same cytopathic effects as in IBRS-2 such as shrinking of cytoplasm, rounding of cells and detachment of cells from the surface of flask within 24 h. For raising of hyperimmune sera, PSV was grown in IBRS-2 cell line up to the required volume and purified by ultracentrifugation. With self-raised hyperimmune sera in laboratory rats, ICC was performed in BHK-21 cells infected with PSV. Positive signals consisted of large granular aggregates of virus in the cytoplasm near the nucleus, suggesting that PSV can infect cell lines other than those of porcine origin.

Pathological and molecular investigation of porcine sapelovirus infection in naturally affected Indian pigs.

The aim of the present study was to pathological and molecular investigation of porcine sapelovirus (PSV) in naturally infected Indian pigs of various age groups. Eight samples (16%) out of 49 necropsied animals were positive for PSV on the basis of pathological and molecular investigation. Major lesions of PSV positive cases were thickening and clouding of meninges, congestion in brain, severe to moderate congestion in lungs along with froathy exudates in trachea, thickening of intestinal mucosa, especially mucosal folds of ileum. Microscopic lesions of PSV positive cases in CNS were perivascular cuffing, neuronophagia and focal gliosis. In lungs, interstitial pneumonia was noticed in all cases, and intestinal lesions comprised of sloughing of villi epithelium, moderate to severe congestion of blood vessels and infiltration of mononuclear cells mainly plasma cells in both large and small intestine. RT-PCR results of total cases examined for PSV were targeted for PSV 3D Polymerase, 5'UTR region and VP1 gene respectively. Genetic characterization was done on the basis of viral capsid protein 1 (VP1) gene of PSV. The sequencing and phylogenetic analysis of amplified VP1 gene product showed maximum identity 85-90% with South Korean, KJ821021.1 and Indian, KY053835.1 strain of PSV. Further explorative surveillance and epidemiological studies are suggested to find out the real impact of this economically important disease affecting pigs population of India.

Porcine sapelovirus among diarrhoeic piglets in India.

Porcine sapelovirus (PSV) A belongs to the genus Sapelovirus, family Picornaviridae. PSV infections in pigs have been reported from European countries, United States, Japan, China, Korea and Brazil. The virus has been isolated/detected from faeces of healthy pigs as well as those affected with diarrhoea, respiratory signs, encephalitis, skin lesions and fertility disorders. This study was planned to investigate whether PSV is prevalent among pigs in India and to characterize PSV encountered in the study population. The study revealed that five of 70 (7.14%) faecal samples were found positive for PSV using RT-PCR. Three viruses were successfully isolated from faecal samples using IB-RS-2 cell line. Complete genome sequencing and analysis of one Indian PSV isolate revealed highest homology (88%) with V13 strain from England. Phylogenetic analysis of the complete polyprotein nucleotide sequences of 14 strains of PSV classified the viruses into four distinct clades. This first report from India adds to our knowledge on genetic diversity of PSV detected so far among pigs in different countries. A large-scale surveillance of the virus is required to understand its genomic diversity and economic impact.

Spatial prediction of landslide susceptibility in parts of Garhwal Himalaya, India, using the weight of evidence modelling.

Garhwal Himalaya in northern India has emerged as one of the most prominent hot spots of landslide occurrences in the Himalaya mainly due to geological causes related to mountain building processes, steep topography and frequent occurrences of extreme precipitation events. As this region has many pilgrimage and tourist centres, it is visited by hundreds of thousands of people every year, and in the recent past, there has been rapid development to provide adequate roads and building infrastructure. Additionally, attempts are also made to harness hydropower by constructing tunnels, dams and reservoirs and thus altering vulnerable slopes at many places. As a result, the overall risk due to landslide hazards has increased many folds and, therefore, an attempt was made to assess landslide susceptibility using 'Weights of Evidence (WofE)', a well-known bivariate statistical modelling technique implemented in a much improved way using remote sensing and Geographic Information System. This methodology has dual advantage as it demonstrates how to derive critical parameters related to geology, geomorphology, slope, land use and most importantly temporal landslide distribution in one of the data scarce region of the world. Secondly, it allows to experiment with various combination of parameters to assess their cumulative effect on landslides. In total, 15 parameters related to geology, geomorphology, terrain, hydrology and anthropogenic factors and 2 different landslide inventories (prior to 2007 and 2008-2011) were prepared from high-resolution Indian remote sensing satellite data (Cartosat-1 and Resourcesat-1) and were validated by field investigation. Several combinations of parameters were carried out using WofE modelling, and finally using best combination of eight parameters, 76.5 % of overall landslides were predicted in 24 % of the total area susceptible to landslide occurrences. The study has highlighted that using such methodology landslide susceptibility assessment can be carried out in vast stretches of Himalaya in short time in order to assess the impact of development as well as climate change/variability. The resultant map can play a critical role in selecting areas for remedial measures for slope stabilisation as well planning for future development of the region.

Injury count model for quantification of risk of occupational injury.

Reduction of risk of occupational injuries is one of the most challenging problems faced by industry. Assessing and comparing risks involved in different jobs is one of the important steps towards reducing injury risk. In this study, a comprehensive scheme is given for assessing and comparing injury risks with the development of injury count model, injury risk model and derived statistics. The hazards present in a work system and the nature of the job carried out by workers are perceived as important drivers of injury potential of a work system. A loglinear model is used to quantify injury counts and the event-tree approach with joint, marginal and conditional probabilities is used to quantify injury risk. A case study was carried out in an underground coal mine. Finally a number of indices are proposed for the case study mine to capture risk of injury in different jobs. The findings of this study will help in designing injury intervention strategies for the mine studied. The job-wise risk profiles will be used to prioritise the jobs for redesign. The absolute indices can be applied for benchmarking job-wise risks and the relative indices can be used for comparing job-wise risks across work systems.

Injury severity assessment for underground coalmine workers.

An injury severity model is proposed for assessment of injury incidents in industrial settings. A classification scheme for injury incidents considering interactions is also developed. The injury severity model considers injury potential in the form of unsafe conditions and analyzes its transfer to actual injury of varying severity. A case study was conducted in an underground coalmine of eastern India. An observed reduction in risk realization is explained through the model. Presence of interactions is found to be the most significant incident attribute of injury occurrences. The classification scheme and the results obtained from this study will help in improving accident/injury investigation reporting and devising preventive measures for reducing injury severity.

Analgesic activity of Amorphophallus campanulatus tuber.

The methanol extract of Amorphophallus campanulatus tuber, given orally at the doses of 250 and 500 mg/kg, showed significant analgesic activity in mice.

Protein A-activated macrophages induce apoptosis in Ehrlich's ascites carcinoma through a nitric oxide-dependent pathway.

Protein A (PA) of Staphylococcus aureus has been demonstrated to possess anti-tumor activity against a wide variety of tumors. In the current study we endeavored to obtain a mechanistic insight into PA-mediated Ehrlich's ascites carcinoma (EAC) killing. Our results indicate that PA stimulates generation of nitric oxide (NO) from murine peritoneal macrophages. Nitric oxide in turn induces cytotoxic damage to the tumor cells. Analysis of the morphological features and cell cycle phase distribution pattern of nuclear DNA revealed an induction of apoptosis (appearance of sub-G0/G1 population) in EAC after PA treatment. We have further elaborated the alterations in the expressions of the proto-oncoproteins p53 and Bax, together with a change in the ratio of Bcl-2/Bax in the treated tumor cells, which favor apoptosis. PA-induced apoptosis and changes in the expression of oncoproteins in the tumor cells was prevented by the suppression of NO release by the addition of L-NAME, the competitive NOS inhibitor, suggesting a possible mechanism by which PA exerts its anti-tumor activities involving nitric oxide through the alteration in the expressions of pro-apoptotic proteins.

Synergistic immunopotentiating effects induced by T-cell and B-cell superantigen in mice.

Staphylococcal Enterotoxin-A(SEA), a 27kDa monomeric protein, produced by some strains of Staphylococcus aureus, is a prototype T-cell superantigen which causes proliferation of cytotoxic T-lymphocytes and produces cytokines like TNF-alpha and IFN-gamma. Recently Protein A (PA), a 42 kDa membrane protein of the Staphylococcus aureus Cowan-I strain, has been termed a B-cell super antigen. It has been shown to cause multiple immunological responses. In the present study we examined the effect of these two superantigens used separately as well as combination in a normal mouse system. It has been shown that combination treatment of PA and SEA is more effective than that of each individual one. FACS analyses of cell cycles showed that a finely turned cellular collaboration occurred in various phases of cell growth and proliferative response compared with controls (P<0.01). It has also been shown that the percentage of various cell types bearing different clusters of differentiation markers, e.g., CD8+, CD34+ increases considerably due to the combined effect of PA and SEA. We also observed that co-administration of both the elicits different soluble mediators like cytokines (TNF-alpha, INF-gamma, IL-1beta). No apoptotic phenomenon was observed (from the cell cycle analysis) for the dose of PA and SEA, used for the experiments, suggesting that these doses of PA and SEA should be non-toxic.

Solution structure of an immunoactive peptide fragment of Staphylococcal protein-A.

Staphylococcal protein-A (SpA) is known to bind the Fc fragment of immunoglobin G in vitro and induce a myriad of immunogenic responses in vivo. The latter is ascribed to be due to the interaction of Fc and SpA. It has also been proposed that in vivo proteolytically cleaved fragments of SpA may be functioning in the same manner. One such fragment (EQQNAFYEILHLPNLNEEQR), fragment 8-27 of the B-domain (SpA-B), was recently shown to exhibit in vivo immunogenic response [Sinha, P., Sengupta, J., and Ray, P. K. (1999) Biochem. Biophys. Res. Commun. 258, 141-147]. As a first step towards understanding the mode of interaction of this peptide with the Fc fragment, we have studied the solution conformation of this isolated peptide by CD and NMR. The peptide, with 7 contact residues in the crystal structure of the SpA-B/Fc complex and comprising of mostly helixI and part of helixII of the 3-helix bundle of SpA-B, was found to be present predominantly in extended structure. However it showed nascent turn/helix like conformations around F14 & Y15. These two residues are known to play a vital role in SpA-B/Fc interaction as deciphered from crystal structure and NMR studies of SpA-B/Fc complex and mutational studies. The implications of our results, especially the nascent conformations found around F14 & Y15, in design of SpA-B mimetic small molecules are discussed.

Protein A-activated rat splenic lymphocyte proliferation involves tyrosine kinase-phospholipase C-protein kinase C pathway.

Protein A (PA) of Staphylococcus aureus was long been known for its affinity towards the Fc domain of immunoglobulin G. It is now well established that PA is a potent biological response modifier showing simultaneously antitumor, antitoxic, and anticarcinogenic properties. This bacterial protein was also observed to stimulate production of cytokines. But the molecular mechanism(s) of immunocyte activation by PA still remained essentially unknown. In this report, we demonstrate a hitherto undescribed role of PA as a signal inducer in rat splenic lymphocytes. Our studies describe that PA induces transition of G0/G1 to S and G2/M phases of cell cycle, thus ultimately stimulating splenic lymphocyte proliferation. It has also been revealed that PA binds to rat splenic lymphocytes in a dose dependent manner and stimulates proliferation via tyrosine kinase-phospholipase C (PLC)-Ca2+-dependent protein kinase C (PKC) pathway. These observations will be of valid help in correlating the immunostimulatory activities of PA with the molecular mechanism(s) of its action.

Protection of apoptotic cell death by protein A.

The word "Apoptosis" or pragrammed cell death is described as the ultimate end of multiple cellular events converging from numerous initiating events to the ultimate death of a cell or organism. Several processes, such as initiation of death signals at the plasma membrane, expression of pro-apoptotic oncoproteins, activation of death proteases, endonucleases etc., that ultimately coalesce to a common irreversible execution phase, lead to cell demise. Counteracting the death signals are cell survival factors. A balance between the cell death and cell survival factors plays a major role in the decision making process as to whether a cell should die or must live. It is, therefore, hypothesized that if the balance can be shifted in favor of cell survival, one might be able to arrest the aging process, save the injured cells or else if the balance is shifted toward cell-kill it might help destroy tumors and other undesirable cells. Protein A (PA) of Staphylococcus aureus has been found to have multifarious biological response modifying properties. It has been shown to possess anti-tumor, antitoxic, anti-parasitic and antifungal activities. It also acts as a potent immunostimulator. PA can protect bone marrow progenitor cells from zidovudin(AZT)-induced apoptosis and can stimulate immunocyte proliferation, thereby helping to replenish/restore the depleted hematopoietic cell pool. Such ability to replenish hematopoietic cells is a common property of PA observed against a number of toxic drugs/chemicals, such as cyclophosphamide, benzene, aflatoxin, salmonella endotoxin, etc. Interestingly, it was further demonstrated in our laboratory that PA can selectively kill tumor cells without affecting normal cells of the host. A search for the mechanisms of PA action revealed that this bacterial protein could shift the balance between pro- and anti-apoptotic proteins in favor of survival in normal cells, but in favor of cell death in tumor cells at a particular dose level. This unique property of PA suggests that controlled use of such type of Biological Response Modifier might help in controlling both cell growth and death phenomena.

Protein-A activates membrane bound multicomponent enzyme complex, NADPH oxidase in human neutrophils.

Protein-A, 42KD cell wall glycoprotein of S. aureus Cowan I enhance mononuclear and polymorphonuclear cell counts in vivo and possesses antitoxic, antitumor, properties. In order to explain the mechanism of its function, the respiratory burst phenomenon in cell and cell free system was studied using lucigenin-dependent chemiluminescence technique. A dose dependent increase in protein A-mediated generation of superoxide radical was observed in resting and PMA stimulated neutrophils. Superoxide dismutase (SOD) was used to confirm the production of superoxide radicals (O2-). To understand the mechanism of protein-A induced O2- generation; NADPH oxidase activity was measured in cell free system using NADPH as a substrate. A significant increase in NADPH oxidase activity was observed in the membrane and post-nuclear supernatant fraction of activated human neutrophils. Cytosolic fraction showed slight enzyme activation. Protein A (SpA)-induced NADPH oxidase activation in the membrane fraction was observed even in the absence of the substrate NADPH. These data indicate that protein A attenuate the NADPH oxidase system to produce O2- radicals.

Mechanisms of protein A superantigen-induced signal transduction for proliferation of mouse B cell.

Apart from many of the biological properties of protein A (PA) of Staphylococcus aureus, it has been recognized recently as a B-cell superantigen. Therefore, we investigated the molecular mechanisms of PA superantigen-induced mice splenic B-cell proliferation. Treatment of resting B cells with PA-evoked cell proliferation. Binding of PA to B cells led to a cascade of signal transduction mechanisms involving tyrosine kinase that activated phospholipase C, which in turn activated protein kinase C (PKC), and translocated it from cytosol to membrane. Mitogen-activated protein (MAP) kinase has been found to be activated down-stream of PKC in this signal pathway, which ultimately caused an activation of serum-responsive factor (SRF). Inhibition at any step of this signaling cascade could block B-cell proliferation. PA could also stimulate the Bcl-2 gene expression at protein level thereby supporting the pro-proliferative effect of PA. Thus, the molecular mechanisms related to PA-induced B cell proliferation has been delineated in this report as tyrosine kinase > PLC > PKC > MAP kinase > SRF > Bcl-2. Knowledge gathered from these observations might be of immense help to study the immune cell proliferation as a part of immunoactivation process. Also, the development of suitable inhibitors of the signaling pathway outlined here might provide clues as to how to abrogate pathologic antibody production in many disease processes.

Protection by protein A of apoptotic cell death caused by anti-AIDS drug zidovudine.

Zidovudine, the anti-AIDS drug, caused inhibition of mitogen-induced proliferation and perturbation of cell-cycle progression of cultured bone marrow cells of mice. There was significant hypoploidy observed in flow cytometric analysis of AZT-treated bone marrow cells. In apo-direct analysis, cells showed apoptosis in G0/G1 phase. In DNA gel analysis, characteristic laddering of apoptosis was observed in AZT-treated bone marrow cells. We demonstrated that, when the animals were pretreated with protein A (PA) of Staphylococcus aureus, the apoptotic changes could be prevented in bone marrow cells of AZT-treated animals. There is a significant (p < 0.05) increase in proliferation of bone marrow cells subjected to mitogen treatment in PA+AZT-treated animals, compared to only AZT-treated animals. However, cell-cycle phase distribution was not hampered and no laddering in DNA gel analysis was also observed in this group. In apo-direct analysis, PA treatment showed significant (p < 0.001) inhibition of AZT-induced apoptosis. These observations indicate that by using a suitable agent such as protein A the toxic side effects of AZT could be minimized.

Stress genes and species survival.

Stress genes can be ascribed to have been generated by the organism for their intrinsic urge to survive against the changing environmental odds, during the evolutionary process. This concept has been supported by a large number of reports describing individual types of phenomena. These have been reconciled and globalised in terms of their relevance in this article. Supporting evidences have been drawn from the literature which indicated that by using different types of inducer one can express heat shock proteins. Similarly, several types of stress inducers, such as calorie restriction, LPS stimulation and Staphylococcal Protein-A stimulation, it was possible to induce a wide array of biological, biochemical and immunological reactions. Such biological reactions rendered protection against toxic, carcinogenic, metabolic, as well as biological stresses induced by microorganisms. Heat shock proteins have been implicated as having a role in providing resistance to the host against different types of stressors. In this article, some mechanistic schemes have been proposed as possible pathways globalising such phenomena. A minute amount of stress inducers has been observed to have helped expression of stress resistance genes, providing increased capability to the host to protect itself against myriads of both biotic and abiotic stressors. More understanding about such phenomena would help in keeping our physiological systems vigilant and our bodies healthy, fighting out the stress-related events effectively.

Glutathione reduces the toxicity associated with antitumor therapy of ascites fluid adsorbed over Staphylococcus aureus Cowan I in tumor bearing mice.

It has been well documented in the literature that the removal of circulatory immune complexes (CICs) from the host circulation leads to the immunopotentiation as well as generation of antitumor responses in a variety of tumors in rats, cats, dogs and human patients. CICs are the major immunosuppressive factors in tumor bearing host. Protein A (PA) has been extensively used for the removal of these CICs from the sera/plasma of tumor bearers, because PA has the ability to bind with the Fc portion of mammalian immunoglobulins. Previously, we reported for the first time a potent antitumor response by the inoculation of cell free Ehrlich's ascites fluid adsorbed in vitro over PA containing Staphylococcus aureus Cowan I (SAC) in Ehrlich's ascites tumor model. However, there was toxicity associated with this form of therapy in terms of early death of treated animals and the depletion of hepatic glutathione pool as well as phase I biotransformation enzyme and increase in glutathione-S-transferase (GST) activities. In the present investigation, tumor bearing animals were treated intraperitoneally (i.p.) on alternate days for 15 days with adsorbed ascites fluid (ad-ASF) (0.1 ml) and glutathione (GSH) (250 mg/kg body weight) separately. We found that GSH supplementation increases mean survival time of GSH and ad-ASF treated mice up to 37.2 days in comparison with 19.9 days for only ad-ASF treated animals, while percent increase in body weight was found to be not affected by the GSH substitution, which remains significantly lower (P < 0.01) in comparison to the tumor control animals. GSH supplementation causes a significant decrease (P < 0.05) of glutathione-S-transferase and restoration of aniline hydroxylase activity (P < 0.05) and aminopyrine-N-demethylase activity. We have also observed that GSH supplementation does not alter the tumor cell viability and tumor cell counts in ad-ASF treated animals in comparison to only ad-ASF treated animals, which indicates that GSH supplementation does not alter the antitumor effect of the therapy. Treatment of Ehrlich's ascites tumor bearing mice with ad-ASF and glutathione increased their survival, but did not reduce the mortality of animals because of tumor.

Protein A of Staphylococcus aureus evokes Th1 type response in mice.

Protein A (PA) of Staphylococcus aureus is known to elicit several cytokines such as IFN gamma, TNF alpha and IL1. However, it has not been delineated yet as to which differentiation pathway lymphocytes follow after stimulation by PA. In this report, we attempted to collect such evidences. Cytokines, such as IFN gamma, IL2, IL4, IL6, IL10, TNF alpha, IL1alpha and IL1beta were measured in serum by ELISA. Our results show that 1 microg dose of PA stimulates the production of IFN gamma (115 +/- 5 pg/ml), TNF alpha (250 +/- 8 pg/ml) and IL1alpha (100 +/- 5 pg/ml) as compared to control levels of, 22 +/- 2, 20 +/- 2 and 35 +/- 3 pg/ml respectively whereas IL2 and IL1beta secretion were less (beyond the lower detection limit of the kit and 25 +/- 1 pg/ml, respectively) as compared to control (28 +/- 2 and 52 +/- 4 pg/ml, respectively). Larger dose of PA (10 microg) increases the expression of IL2 (75 +/- 3 pg/ml), TNF alpha (1380 +/- 120 pg/ml), IL1alpha (495 +/- 10 pg/ml) and IL1beta (110 +/- 7 pg/ml) as compared to controls described above. We also observed that 1 microg dose of PA decreases IL4, IL6 and IL10 secretion to 9 +/- 1, 10 +/- 1 and 10 +/- 2 pg/ml, respectively, whereas 10 microg dose also decreased them to 11 +/- 1, 12 +/- 2 and 30 +/- 4 pg/ml, respectively as compared to the background controls, i.e. 50 +/- 5, 50 +/- 2 and 215 +/- 9 pg/ml respectively. The ratio of IFN gamma to IL4 increased and the peak value at 4 h, came to 13 +/- 1 and 9.6 +/- 0.5 with 1 microg and 10 microg PA, respectively, which is an established parameter indicating a Th1 type response. Flow cytometry analysis of CD4+/CD8+ cells, and c-myc protein expression by splenocytes indicate that 1 microg dose of PA causes 2-fold increase of CD4+ cells with no change in CD8+ cells, and 10-fold increase in c-myc protein, whereas 10 microg dose increases CD4+ cells 4-fold, CD8+ cells 3-fold and c-myc protein 100-fold. The cell cycle data shows an induction of apoptosis in thymocytes and splenocytes with the large dose (10 microg), whereas the 1 microg dose does not show any apoptosis. This report indicates that a Th1 response is induced in mice, after PA inoculation at a dose of 1 microg animal. Thus, cytokine mediated therapeutic strategies should consider the fact that an induction of large concentration of some cytokines might become detrimental to the host.