Loading

Clinical Research in Infectious Diseases

Action of Synthetic Peptide LKEKK in Experimental Tuberculosis

Research Article | Open Access | Volume 8 | Issue 1

  • 1. Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Science Avenue, Russia
  • 2. Laboratory of Peptide Chemistry, State Research Institute of Highly Pure Biopreparations, Saint Petersburg, Russia
+ Show More - Show Less
Corresponding Authors
Elena V Navolotskaya, Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Science Avenue, Russia
Abstract

In the present study, we investigated the activity of the synthetic peptide LKEKK (Np5) in murine model of tuberculosis induced by Mycobacterium bivisbovinus 8 strain. Therapy with Np5 at doses of 0.01, 0.1, and 1µg/kg (5 daily injections) decreased the lung damage index compared to untreated controls and to those treated with isoniazid alone. The growth of M. bivis-bovinus 8 in spleen culture was decreased. Study of cytokine production showed that on the 24th day after treatment with Np5 (doses of 0.01, 0.1, 1µg/kg) the secretion of IL-2 was restored to the level seen in uninfected animals. IFN-γ production be both thymus and spleen cells, as well as its circulating levels in serum, was increased by the Np5 treatment. Concurrently, IL-4 production was decreased in the same cell types and in the serum. The Np5 treatment also stimulated the macrophage functions, which had been decreased by tuberculosis infection and isoniazid therapy, with an improved phagocytosis activity of peritoneal macrophages. Thus, the Np5 treatment increased the efficacy of anti-tuberculosis therapy as well as strength of the immune response.

Keywords

• Protein

• Peptide

• Receptor

• Cytokine

• Tuberculosis

CITATION

Navolotskaya EV, Zinchenko DV, Kolobov AA, Murashev AN (2024) Action of Synthetic Peptide LKEKK in Experimental Tuberculosis. Clin Res Infect Dis 8(1): 1063.

INTRODUCTION

Tuberculosis is a widespread in the world chronic infectious disease of humans and animals, which is caused by various types of mycobacteria from the Mycobacterium tuberculosis complex group (M. tuberculosis, M. bovis, M. africanum, M. microti, M. canettii); it causes more than 1.5 million deaths annually [1-3]. Experts note that over the past few decades, drug resistance of mycobacterial strains has increased significantly. Currently, patients are increasingly becoming infected with strains of mycobacteria that are resistant to almost all antibiotics, that requires an urgent search for new drugs and treatment methods [4].

A unique feature of mycobacteria is the complexity of its cell wall. This structure consists of a plasma membrane, a cell wall core and an outer envelope, including many complex lipids, peptidoglycans and mycoic acids [5-8]. Mycolic acids, long- chain branched fatty acids, containing 60-90 carbon atoms per molecule, are an exclusive component of the cell wall of mycobacteria; they make the surface of the bacilli waxy and highly hydrophobic, providing protection against hydrophilic antibiotics, oxidative damage and the host immune response [9].

Several   years   ago,   we   synthesized   the   peptide   LKEKK corresponding to the sequences 16-20 of human thyroxin-α1 (TM-α1) and 131-135 of interferon-α2 (IFN-α2) and showed that it binds with high affinity to murine macrophage-like cells of line RAW 264.7. In the 10-1000 nM concentration range, the peptide dose-dependently increased the Nitric Oxide (NO) production, the activity of soluble guanylate cyclase (sGC), as well as the adhesion, spreading, and capacity to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro by the cells. The synthetic peptide with inverted KKEKL sequence tested in parallel was inactive. Thus, the peptide LKEKK binding to RAW 264.7 cells leads to an increase in NO-synthase, guanylate cyclase and phagocytic activity [10]. The purpose of this work is to study the effect of the peptide LKEKK (Np5) on a mouse model of tuberculosis.

MATERIALS AND METHODS

Chemicals

IL-2, IL-4, IFN-γ and other chemicals were obtained from Sigma (St. Louis, MO).

Peptides

Peptide LKEKK (Np5) was synthesized on   an   Applied Bio systems Model 430A automatic synthesizer (USA) using the Boc/Bzl tactics of peptide chain elongation as described previously [11]. The peptide was purified to homogeneous state by preparative reverse-phase HPLC (Gilson chromatograph, France) on a Delta Pack C18 column, 100A (39×150 mm, mesh size 5µm; flow rate 10ml/min, elution with 0.1%TFA, gradient of acetonitrile 10-40% in 30min). The molecular masse of peptide was determined by fast atom bombardment mass spectrometric analysis (Finnigan mass spectrometer, San Jose, CA). The data of amino acid analysis (hydrolysis by 6 M HCl, 22h, 110°C; LKB 4151 Alpha Plus amino acid analyzer, Sweden) and mass spectrum analysis are presented in (Table 1).

Animal Infection and Treatment

Infection of 200 white wild type mice, obtained from Lab. Animal Nursery, Rappolovo, Russia, was performed with disseminated tuberculosis (TB) by injection of M. bovis-bovinus 8 suspension (0.1mg in 0.2mL of saline, contained 108 bacterial bodies). Two mice were sacrificed every two days from day 7 after the inoculation, and lungs were inspected. When single subsidiary foci (< 1 mm) in the lungs were seen in the sacrificed mouse (day 12 after inoculation), all other animals were selected to one of the groups and isoniazid therapy (at a sub-therapeutic dose of 10 daily, subcutaneous) was started. Np5 treatment consisting of 5 daily intraperitoneal (i.p.) injections of doses of 0.01, 0.1, 1.0 and 10µg/kg, started on day 20 after inoculation (when multiple subsidiary foci were found on the autopsy of untreated mice). One group of animals was treated with a second course of Np5 treatment at a dose of 1µg/kg, beginning 2 days after the first treatment ended (day 26). Control groups included mace without therapy (inoculation control) and mice on isoniazid therapy alone (therapy control). Samples were harvested on day 4, 10, 17 and d 24 after the end of 5 five days course of Np5 therapy, corresponding to the treatment days 28, 34, 41 and 48. At least 5 mice from each group were examined.

Severity of Experimental TB

Severity of experimental TB was evaluated by visual examination and calculated as a “Lung Damage Index”. The following criteria were used single submiliary foci were estimated at 0.5 units (U), multiple submiliary foci (< 20) as 1.0 U, multiple submiliary foci (> 20) as 1.5 U, single miliary foci as 1.75 U, multiple associated submiliary and single military foci as 2.0 U, military foci (< 10) as 2.25 U, multiple associated military foci as 2.75 U, small caseous foci as 3.0 U, disseminating caseous as 4.0 U, damage to the entire lung as 5.0 U. In the case of lung maceration by serous liquid, the index was increased (by 0.25 1.0 U), depending on the extent of damage. Lung and spleen weight were also measured and compared to the total weight of the mouse to provide an organ weight index.

Mycobacterium Contamination

Mycobacterial contamination was assessed by bacteriological investigation of spleen tissue homogenate cultured on solid egg Lowenstein-Jensen media with the growth density of M. bovis- bovinus 8 expressed in Colony Forming Units (CFU). Colony count was performed by visual examination of solid media surface [12]. If number of colonies was countable (< 100) exact count was performed. Many conjugated colonies (but not solid growth) were counted as 200. Solid mycobacterial growth was counted as approximately 300 colonies. Two cultures for each sample were performed and then median was counted. Data were presented as median (min-max). We estimated mycobacterial contamination be spleen examination because clearance in spleen was going more quickly the in lunges.

Peritoneal Macrophage Activity

Phagocytosis was studied in peritoneal macrophages in cell culture. Cells were plated at a concentration of 106 cells per Petri dish, and media was added that contained 107 Saccharomyces cerevisiae cells, opsonized by mouse serum. Phagocytic activity, the percent macrophages involved in phagocytosis, and phagocytic digestion, the number of yeast digested be macrophages after 1.5 h of incubation, were counted by microscopy.

Cytokine Production and Cytokine ELISAs

For cytokine determination, spleen cell suspensions were diluted to a concentration of 107/mL in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 1 mM PMSF, 10μg/ml aprotinin, 10μg/ml leupeptin, and 10μg/ml pepstatin A. 100 µl of cell suspension was added to each well of 96-well cell culture plates. RPMI 1640 alone was added to control wells. Cytokine production was induced by concanavalin A (Con A, final concentration of 2.5µg/ml). Cells were incubated for 24 h at 37°C in a humidified atmosphere 5% CO2. After incubation 150 µl of supernatant was removed from each well. Supernatants were stored at-70°C. Concentrations of Il-2, Il-4 and IFN-γ in cell supernatants were measured by ELISA kits. Results are expressed as U/ml. ELISAs were carried out using according to the manufacturer’s instructions (BD Biosciences, San Jose, CA). Data are presented as mean ± SEM.

Statistical Analysis

The data were evaluated using the Mann-Whitney test. The results are presented as mean ± SEM or as median (min-max).

RESULTS

Peptides

The main characteristics of the peptide LKEKK (Np5) (purity, amino acid content, and molecular mass) are shown in (Table 1).

Table 1: Main characteristics of the peptides.

Peptide

Purity, %

Amino acid analysis data

Molecular mass, D

Np5

> 98

Glu 1.08, Leu 1.00, Lys

3.32

645.4

(calculated value - 644.87)

Severity of Experimental TB

Therapy with Np5 rapidly changed the progression of TB infection in mice: at all doses, the peptide decreased the lung damage index when measured 4 days after the end of 5 days of the therapy (day 28 of the infection). Significant differences were seen between the animals treated with Np5 doses of 0.01, 0.1, 1 µg/kg and untreated controls (1.62 ± 0.37, 1.81 ± 0.12 and 1.97 ± 0.28 compared to 2.73 ± 0.15, respectively, p < 0.05). A significant difference was also seen between animals treated with a dose of 0.1µg/kg and those treated with isoniazid alone: on day 24 after the end of 5 days of the Np5 therapy, the lung damage index was also significantly lower at Np5 doses of 1.0, 0.1, 0.01µg/kg (2.56± 0.15, 2.58 ± 0.14 and 2.60 ± 0.16 compared to 3.32 ± 0.26 in the control group, respectively, p < 0.05).

At the dose 0.1 µg/kg other beneficial effects of Np5 were also seen. There were significant increases in body weight (30.7% vs. 20.5% in isoniazid treated mice, 24 days after therapy) and decreases in lung weight index (1.37 ± 0.06 vs. 1.97 ± 0.28, p < 0.05) and spleen weight index (1.59 ± 0.25 vs. 1.94 ± 0.32), There was also a significant decrease in the growth of M. bovis-bovinus 8 cultured from spleen: 200 (200-200) CFU vs. 275 (250-300) CFU in isoniazid treated mice, p < 0.05.

Cytokine Production

A decrease of production of IL-2 in Con A-stimulated spleen cells was seen after TB infection with extensive lung damage (Table 2). The Np5 treatment led to an increase in IL-2 production in all treated groups, at all-time points after the therapy. IL-2 production in animals treated with Np5 doses of 0.1 and 0.01 µg/ kg was markedly increases in comparison to those treated with isoniazid alone (41.6 ± 5.6 U/ml and 39 ± 2.8 U/ml vs. 25.2 ± 2.4 U/ml, respectively) as early as 4 days after Np5 therapy. Twenty- four days after the start of therapy, the IL-2 level in animals treated with Np5 doses of 0.1 or 1µg/kg (10 injections) returned to the level of uninfected mice.

Table 2: Effect of peptide LKEKK (Np5) on Con A-induced production of IL-21, IL- 42, IFN-γ2 by spleen cells.

Treatment

Cytokine (U/ml ± SEM)

IL-2

IFN-γ

IL-4

Intact mice

74.3 ± 6.5

22.5 ± 2.4

11.2 ± 1.6

Infected mice

12.5 ± 1.6*

10.3 ± 1.4

20.2 ± 2.6

Isoniazid + Np5 (0.01 µg/kg)

39.0 ± 2.8*

12.5 ± 1.7

17.1 ± 1.9*

Isoniazid + Np5 (0.1 µg/kg)

41.6 ± 5.6*

13.7 ± 1.9*

15.7 ± 2.1*

Isoniazid + Np5 (1.0 µg/kg)

45.8 ± 5.0*

14.9 ± 1.5*

14.3 ± 2.0*

Isoniazid + Np5 (10 µg/kg)

53.5 ± 6.2*

18.6 ± 2.0*

13.2 ± 1.4*

Isoniazid treated

25.2 ± 2.4

11.7 ± 1.3

19.0 ± 2.2

*Significant difference from the therapy with isoniazid alone (p < 0.05). 1The evaluation was performed 10 days after the end of Np5 treatment. 2The evaluation was performed 17 days after the end of Np5 treatment.

Basal IFN-γ production in spleen cells was not different from isoniazid-treated control mice as determined 4 days after Np5 therapy. Ten days after the therapy, however, there was a significant elevation of basal IFN-γ production in animals treated with Np5 at doses of 0.1 and 1µg/kg i.p. (5 or 10 injections), and by 17th day this increase was seen in all Np5 treated animals. No significant changes in basal Il-4 production were seen after treatment with Np5.

Con A-stimulated production of IFN-γ and Il-4 in spleen cells was significantly affected by Np5 treatment (Table 2). It is interesting to note that changes in the production of Il-4 and IFN-γ were opposite in direction: the IFN-γ production was increased, whereas the Il-4 production was decreased.

Peritoneal Phagocytes Function

Extensive and wide spread lung damage with TB lead to a decrease in the activity of the peritoneal phagocytes and to a poor phagocytic digestion of yeast cells. The average phagocytic activity 28 days after infection was 4.6% compared to 64.2% 9n uninfected mice (p < 0.01). Phagocytic digestion decreased similarly. Isoniazid therapy caused an increase in the parameters of the phagocytic activity, but not to the level of uninfected mice. When measured 4 days after the treatment (Table 3), Np5 therapy markedly elevated phagocytic activity to 38.5% after a dose of 0.1µg/kg, compared to 19.4% in the isoniazid control group (p < 0.05). Np5 treatment also increased phagocytic digestion (Table 3): 228 (83-435) yeast killed per 1.5 h vs. 173 (139-319) in intact mice, (p < 0.05).

Table 3: Phagocytic activity of murine peritoneal macrophages, day 4.

Treatment

Phagocytic macrophages (%)

Digestion (number of digested yeast during 1.5 h)

Intact mice

64.2 (61.0-66.0)

173 (139-319)

Infected mice

4.6 ((2.0-8.0), *p < 0.01

6 (0.0-34.0), *p < 0.01

Isoniazid treated

19.4 (6.0-34.0), *p < 0.01

58 ((17.0-147.0), *p < 0.05

Isoniazid + Np5 (0.01 µg/kg)

21.6 (4.0-54.0), *p < 0.01

80 (21.0-215)

Isoniazid + Np5 (0.1 µg/kg)

38.8 (16.0-57.0),

**p < 0.05

228 ((93.0-435.0), **p < 0.01

Isoniazid + Np5 (1.0 µg/kg)

29.8 (11.0-46.0), *p < 0.01

184 ((18.0-306)

Isoniazid + Np5 (10 µg/kg)

38.6 (5.0-62.0), *p < 0.05

246 (8.0-276.0)

Data is presented as median (min-max); *significant difference with uninfected mice; ** significant difference with inoinazid alone.

Ten days after Np5 therapy, we observed a weakening of the drug effect, perhaps because of improvement due to the isoniazid therapy. In fact, 24 days after the treatment the phagocytic activity in the isoniazid treatment group had returned to the level of uninfected mice. However, phagocytic digestion was still depressed: 60 (21-107) vs. 138 (63-290) in uninfected mice, p < 0.05. Np5 treatment, particularly at 0.1µg/kg and 1.0 µg/kg (10 injections), significantly improved the digestion: 199.5 (86-375) and 167 (64-291), respectively, vs. 60 (21-107) in isoniazid alone treated mice, p < 0.05.

DISCUSSION

Despite growing global efforts to eradicate tuberculosis, it killed a total of about 5 million people between 2021 and 2023, and was in fact the second biggest infectious killer after COVID-19. Thanks to vaccination and control of the pandemic, tuberculosis is likely to once again become the leading cause of death in the world, especially since during the COVID-19 pandemic, all health resources were devoted to containing SARS-Cov2 and important anti-tuberculosis programs were ignored. In addition, it should be noted that drug-resistant tuberculosis is currently becoming a serious problem: its treatment is difficult, time-consuming and expensive and often requires the use of toxic and poorly tolerated drugs [13]. Compared to existing anti-tuberculosis drugs, peptides have a number of advantages. First, they have a broad spectrum of activity against various strains of M. tuberculosis, including drug-resistant strains, making them potential candidates for the treatment of drug-resistant tuberculosis. Secondly, peptides are fast acting, which means they can quickly kill germs, which will shorten the duration of treatment. Third, peptides have a low risk of developing drug resistance because they simultaneously target multiple components of bacterial cells and host cells. Finally, peptides are well tolerated by the body. These advantages make potential anti-tuberculosis peptide drugs an attractive option for the development of new treatments for tuberculosis [4].

The understanding of the innunopathogenesis of the TB infection has significantly increased during the last several years. It has become evident that the imbalance in the functional activity of Th1 and Th2 cells plays a key role in the progression of TB [14-16] and correlates with the severity of the diseases. This imbalance is accompanied with a decrease in IL-2, IL-12 and IFN-γ production and impairment in the expression of their receptors [17]. The adequate immune response to Mycobacterium appears to be characterized by greater Th1 activity and production of IFN-γ and IL-2, while a low resistance to infection results from Th2 stimulation and IL-4 production [18-20]. In mice, experimental TB infection is more benign in strains that produce high levels of Th1 cytokines, while mouse strains with lower Th1 cytokines levels are more susceptible [21,22]. Moreover, reactivation of latent TB is accompanied by an elevation of the Th2 cytokine production and by a simultaneous decline in Th1 functions [19,22], in addition, an evidence of Th1 lymphocyte suppression has been reported [23-25].

The use of immune-modulating cytokines for therapy of TB has recently been investigated. Recombinant IL-2 (rIL-2) normalized IL-2 receptor expression, lymphocyte proliferation and, partially, IFN-γ production in lymphocyte cultures obtained from patients with active TB [23,26-27] and a positive effect of rIL-2 has been reported in treatment of murine experimental TB induced by M. avium [28], rIL-2 therapy decreased the number of Mycobacterial CFU in organ cultures, and increased endogenous IL-2 production and IL-2 receptor expression in M. tuberculosis H37RV infected mice [29]. In patients with severe TB that were infected multi-resistant M. tuberculosis strains, rIL-2 led to clinical improvement and an increase in lymphocyte functional activity [30]. IFN-γ used in similar patients decreased M. tuberculosis expectoration and cavity size and increased BCG ingesting and killing by alveolar macrophages [31-34]. In vitro data suggest that IFN-γ inhibits growth of M. avium inside blood monocytes [35]. Finally,  a beneficial effect of IFN-γ on M. tuberculosis clearance was found in multiple drug resistant patients [36]. IL-12 efficacy has been observed in mice and in patients [37-38].

Our results demonstrate that the Np5 treatment increases the production of Th1 cytokines in experimental murine TB, similarly to the interleukins described above. We used M. bovis- bovinus 8 strain with virulence in mice that was similar to M. tuberculosis strains (submiliary foci appeared on day 12 after inoculation and mortality in untreated mice was observed on day 34-46). The model with relatively slow development of TB was used for a better detection of Np5 effects. Other groups of investigators have also used animal models of TB with M. bovis [39-42]. Production of IL-2 by spleen cells was decreased in mice with an extensive TB infection. Other reports also described decreased IL-2 levels in animals and humans with TB [24, 43-44]. In our experiments, IL-2 had significantly less reduction after the treatment with Np5. On the 24th day after the treatment with Np5 production of IL-2 was restored to the level found in the uninfected animals. Production of basal an stimulated IFN-γ by both thymus an spleen cells, as well as circulating serum levels of this cytokine, was increased after the Np5 treatment. At the same time, the IL-2 production by the same cell and in the serum was decreased. These changes, the increase in the IFN-γ production and decrease in IL-4 secretion, suggest that Np5 is elaborating a shit of T helper cell activity towards a Th1-like immune response. Other immune parameters were improved as well - an increase in Con A-stimulated thymic cell proliferation was observed as early as 4 days after the Np5 treatment. Twenty-four days after treatment the proliferative responses for both thymic and spleen cells were restored nearly to the parameters seen in the uninfected animals.

Yeast phagocytosis is a sensitive marker of macrophage function during experimental TB. Ingestion and digestion functions were decreased in inoculated untreated and isoniazid treated animals with an extensive lung damage. Either inhibition of macrophage functions is thought to result from the Mycobacterium [45-47] or from certain anti-TB drugs [48], Np5 treatment actually stimulated the macrophage functions, which had been decreased by TB infection and isoniazid therapy, with an improvement seen in peritoneal macrophage ingesting and digestion ability.

The results presented here suggest that Np5 treatment during isoniazid therapy of TB increases the effectiveness of anti-TB therapy as well as the strength of the immune response. In this study, the treatment with Np5 provided as 5 daily i.p. injections, significantly decreased the lung weight index, the lung damage index, the severity of lung tissue damage, the markers of alteration, and protected the multilayer bronchial epithelium. The growth of M. bovis-bovinus 8 in spleen culture was also decreased.

CONCLUSION

Peptide Np5 with simple structure LKEKK has significant anti-TB activity and is suitable as a basis for the development of complex anti-TB therapy.

DECLARATION OF INTEREST

There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

FUNDING

This study was funded by Fundamental Research Program of the Presidium of RAS “Molecular and Cell Biology” (Grant # 0101- 2014-0086).

REFERENCES
  1. Churchyard G, Kim P, Shah NS, Rustomjee R, Gandhi N, Mathema B, et al. What We Know About Tuberculosis Transmission: An Overview. J Infect Dis. 2017; 216: S629-S635. doi: 10.1093/infdis/jix362. PMID: 29112747; PMCID: PMC5791742.
  2. Furin J, Cox H, Pai M. Tuberculosis. Lancet. 2019; 393: 1642-1656.
  3. Natarajan A, Beena PM, Devnikar AV, Mali S. A systemic review on tuberculosis. Indian J Tuberc. 2020; 67: 295-311. doi: 10.1016/j. ijtb.2020.02.005. Epub 2020 Feb 28. PMID: 32825856.
  4. Jacobo-Delgado YM, Rodríguez-Carlos A, Serrano CJ, Rivas- Santiago B. Mycobacterium tuberculosis cell-wall and antimicrobial peptides: a mission impossible? Front Immunol. 2023; 14: 1194923. doi: 10.3389/fimmu.2023.1194923. PMID: 37266428; PMCID: PMC10230078.
  5. Grzegorzewicz AE, de Sousa-d’Auria C, McNeil MR, Huc-Claustre E, Jones V, Petit C, et al. Assembling of the Mycobacterium tuberculosis Cell Wall Core. J Biol Chem. 2016; 291: 18867-18879. doi: 10.1074/ jbc.M116.739227. Epub 2016 Jul 14. PMID: 27417139; PMCID: PMC5009262.
  6. Chiaradia L, Lefebvre C, Parra J, Marcoux J, Burlet-Schiltz O, Etienne G, et al. Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane. Sci Rep. 2017; 7(1), 12807.
  7. Singh P, Rameshwaram NR, Ghosh S, Mukhopadhyay S. Cell envelope lipids in the pathophysiology of Mycobacterium tuberculosis. Future Microbiol. 2018; 13: 689-710. doi: 10.2217/fmb-2017-0135. Epub 2018 May 17. PMID: 29771143.
  8. Stokas H, Rhodes HL, Purdy GE. Modulation of the M. tuberculosis cell envelope between replicating and non-replicating persistent bacteria. Tuberculosis (Edinb). 2020; 125: 102007. doi: 10.1016/j. tube.2020.102007. Epub 2020 Oct 5. PMID: 33035766; PMCID: PMC7704923.
  9. Singh G, Kumar A, Maan P, Kaur J. Cell Wall Associated Factors of Mycobacterium tuberculosis as Major Virulence Determinants: Current Perspectives in Drugs Discovery and Design. Curr Drug Targets. 2017; 18: 1904-1918. doi: 10.2174/138945011866617071 1150034. PMID: 28699515.
  10. Navolotskaya EV, Sadovnikov VB, Zinchenko DV, Vladimirova VI, Zolotarev YA, Lipkind VM, et al. Effect of the B Subunit of the Cholera Toxin on the Raw 264.7 Murine Macrophage-Like Cell Line. Russ J Bioorgan Chem. 2019; 45: 122-128.
  11. Schnölzer M, Alewood P, Jones A, Alewood D, Kent SB. In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int J Pept Protein Res. 1992; 40: 180-193. doi: 10.1111/j.1399-3011.1992.tb00291.x. PMID: 1478777.
  12. Lecoeur HF, Lagrange PH, Truffot-Pernot C, Gheorghiu M, Grosset J. Relapses after stopping chemotherapy for experimental tuberculosis in genetically resistant and susceptible strains of mice. Clin Exp Immunol. 1989; 76: 458-462. PMID: 2502336; PMCID: PMC1541904.
  13. Khadela A, Chavda VP, Postwala H, Shah Y, Mistry P, Apostolopoulos V. Epigenetics in Tuberculosis: Immunomodulation of Host Immune Response. Vaccines (Basel). 2022; 10: 1740. doi: 10.3390/ vaccines10101740. PMID: 36298605; PMCID: PMC9611989.
  14. Kaufmann SH, Ladel CH, Flesch IE. T cells and cytokines in intracellular bacterial infections: experiences with Mycobacterium bovis BCG. Ciba Found Symp. 1995; 195: 123-132; discussion 132-6. doi: 10.1002/9780470514849.ch9. PMID: 8724834.
  15. Mustafa T, Phyu S, Nilsen R, Jonsson R, Bjune G. In situ expression of cytokines and cellular phenotypes in the lungs of mice with slowly progressive primary tuberculosis. Scand J Immunol. 2000; 51: 548- 556. doi: 10.1046/j.1365-3083.2000.00721.x. PMID: 10849364.
  16. Vasiliu A, Martinez L, Gupta RK, Hamada Y, Ness T, Kay A, et al. Tuberculosis prevention: current strategies and future directions. Clin Microbiol Infect. 2023: S1198-743X (23)00533-5. doi: 10.1016/j. cmi.2023.10.023. Epub ahead of print. PMID: 37918510.
  17. Lange C, Aaby P, Behr MA, Donald PR, Kaufmann SHE, Netea MG, et al. 100 years of Mycobacterium bovis bacille Calmette-Guérin. Lancet Infect Dis. 2022; 22: 2-12. doi: 10.1016/S1473-3099(21)00403-5. Epub 2021 Sep 7. PMID: 34506734.
  18. Balikó Z, Szereday L, Szekeres-Bartho J. Th2 biased immune response in cases with active Mycobacterium tuberculosis infection and tuberculin anergy. FEMS Immunol Med Microbiol. 1998; 22: 199-204. doi: 10.1111/j.1574-695X.1998.tb01207.x. PMID: 9848680.
  19. Dieli F, Singh M, Spallek R, Romano A, Titone L, Sireci G, et al. Change of Th0 to Th1 cell-cytokine profile following tuberculosis chemotherapy. Scand J Immunol. 2000; 52: 96-102. doi: 10.1046/j.1365-3083.2000.00744.x. PMID: 10886789.
  20. Tamburini B, Badami GD, Azgomi MS, Dieli F, La Manna MP, Caccamo N. Role of hematopoietic cells in Mycobacterium tuberculosis infection. Tuberculosis (Edinb). 2021; 130: 102109. doi: 10.1016/j. tube.2021.102109. Epub 2021 Jul 21. PMID: 34315045.
  21. Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, Bloom BR. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J Exp Med. 1993; 178: 2249-2254. doi: 10.1084/jem.178.6.2249. PMID: 7504064; PMCID: PMC2191274.
  22. Actor JK, Olsen M, Jagannath C, Hunter RL. Relationship of survival, organism containment, and granuloma formation in acute murine tuberculosis. J Interferon Cytokine Res. 1999; 19: 1183-1193. doi: 10.1089/107999099313136. PMID: 10547159.
  23. Shiratsuchi H, Okuda Y, Tsuyuguchi I. Recombinant human interleukin-2 reverses in vitro-deficient cell-mediated immune responses to tuberculin purified protein derivative by lymphocytes of tuberculous patients. Infect Immun. 1987;   55:   2126-2131. doi: 10.1128/iai.55.9.2126-2131.1987. PMID: 3114146; PMCID: PMC260667.
  24. Ellner JJ. Regulation of the human immune response during tuberculosis. J Lab Clin Med. 1997; 130: 469-475. doi: 10.1016/ s0022-2143(97)90123-2. PMID: 9390634.
  25. Hirsch CS, Toossi Z, Othieno C, Johnson JL, Schwander SK, Robertson S, et al. Depressed T-cell interferon-gamma responses in pulmonary tuberculosis: analysis of underlying mechanisms and modulation with therapy. J Infect Dis. 1999; 180: 2069-2073. doi: 10.1086/315114. PMID: 10558973.
  26. McDyer JF, Hackley MN, Walsh TE, Cook JL, Seder RA. Patients with multidrug-resistant tuberculosis with low CD4+ T cell counts have impaired Th1 responses. J Immunol. 1997; 158: 492-500. PMID: 8977227.
  27. McDyer JF, Li Z, John S, Yu X, Wu CY, Ragheb JA. IL-2 receptor blockade inhibits late, but not early, IFN-gamma and CD40 ligand expression in human T cells: disruption of both IL-12-dependent and -independent pathways of IFN-gamma production. J Immunol. 2002; 169: 2736- 2746. doi: 10.4049/jimmunol.169.5.2736. PMID: 12193748.
  28. Bermudez LE, Stevens P, Kolonoski P, Wu M, Young LS. Treatment of experimental disseminated Mycobacterium avium complex infection in mice with recombinant IL-2 and tumor necrosis factor. J Immunol. 1989; 143: 2996-3000. PMID: 2553816.
  29. Denis M. Interferon-gamma-treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates. Cell Immunol. 1991; 132: 150-157. doi: 10.1016/0008- 8749(91)90014-3. PMID: 1905984.
  30. Suárez-Méndez R, García-García I, Fernández-Olivera N, Valdés- Quintana M, Milanés-Virelles MT, Carbonell D, et al. Adjuvant interferon gamma in patients with drug - resistant pulmonary tuberculosis: a pilot study. BMC Infect Dis. 2004; 4: 44. doi: 10.1186/1471-2334-4-44. PMID: 15500691; PMCID: PMC529257.
  31. Shimokata K. [Commemorative lecture of receiving Imamura Memorial Prize. Analysis of cellular immunity against tuberculosis in man with special reference to tuberculous pleurisy and cytokines]. Kekkaku. 1996; 71: 591-596. Japanese. PMID: 8936994.
  32. Condos R, Rom WN, Schluger NW. Treatment of multidrug-resistant pulmonary tuberculosis with interferon-gamma via aerosol. Lancet. 1997; 349: 1513-1515. doi: 10.1016/S0140-6736(96)12273-X. PMID: 9167461.
  33. Fenhalls G, Wong A, Bezuidenhout J, van Helden P, Bardin P, Lukey PT. In situ production of gamma interferon, interleukin-4, and tumor necrosis factor alpha mRNA in human lung tuberculous granulomas. Infect Immun. 2000; 68: 2827-2836. doi: 10.1128/IAI.68.5.2827- 2836.2000. PMID: 10768979; PMCID: PMC97494.
  34. Zhuang L, Yang L, Li L, Ye Z, Gong W. Mycobacterium tuberculosis: immune response, biomarkers, and therapeutic intervention. MedComm (2020). 2024; 5: e419. doi: 10.1002/mco2.419. PMID: 38188605; PMCID: PMC10771061.
  35. Shiratsuchi H, Johnson JL, Ellner JJ. Bidirectional effects of cytokines on the growth of Mycobacterium avium within human monocytes. J Immunol. 1991; 146: 3165-3170. PMID: 1901893.
  36. Giosuè S, Casarini M, Ameglio F, Zangrilli P, Palla M, Altieri AM, et al. Aerosolized interferon-alpha treatment in patients with multi-drug- resistant pulmonary tuberculosis. Eur Cytokine Netw. 2000; 11: 99- 104. PMID: 10705306.
  37. Kobayashi K, Kasama T. [The cytokine network and development of immune-based intervention in mycobacterial infection]. Nihon Hansenbyo Gakkai Zasshi. 2000; 69: 77-82. Japanese. doi: 10.5025/ hansen.69.77. PMID: 10979273.
  38. Greinert U, Ernst M, Schlaak M, Entzian P. Interleukin-12 as successful adjuvant in tuberculosis treatment. Eur Respir J. 2001; 17: 1049- 1051. doi: 10.1183/09031936.01.17510490. PMID: 11488308.
  39. Kondo E, Kanai K. Effects of cortisone-treatment on experimental mouse infection with pathogenic and saprophytic Mycobacteria. Jpn J Med Sci Biol. 1977; 30: 209-213. doi: 10.7883/yoken1952.30.209. PMID: 409870.
  40. Kanai K, Kondo E, Yasuda T. An electron microscopy study of intra- cellular mycobacteria in experimental mouse tuberculosis. Tubercle. 1981; 62: 187-195. doi: 10.1016/0041-3879(81)90005-2. PMID: 7032019.
  41. Dahl JL. Electron microscopy analysis of Mycobacterium tuberculosis cell division. FEMS Microbiol Lett. 2004; 240: 15-20. doi: 10.1016/j. femsle.2004.09.004. PMID: 15500974.
  42. Hayashi D, Takii T, Mukai T, Makino M, Yasuda E, Horita Y, et al.   Biochemical   characteristics   among   Mycobacterium   bovis BCG substrains. FEMS Microbiol Lett. 2010; 306: 103-109. doi: 10.1111/j.1574-6968.2010.01947.x. PMID: 20529131.
  43. Estrada García I, Hernández Pando R, Ivanyi J. Editorial: Advances in Immunotherapeutic Approaches to Tuberculosis. Front Immunol. 2021; 12: 684200. doi: 10.3389/fimmu.2021.684200. PMID: 33968090; PMCID: PMC8100343.
  44. Torres-Juarez F, Trejo-Martínez LA, Layseca-Espinosa E, Leon- Contreras JC, Enciso-Moreno JA, Hernandez-Pando R, et al. Platelets immune response against Mycobacterium tuberculosis infection. Microb Pathog. 2021; 153: 104768. doi: 10.1016/j. micpath.2021.104768. Epub 2021 Jan 29. PMID: 33524564.
  45. Phyu S, Tadesse A, Mustafa T, Tadesse S, Jonsson R, Bjune G. Diversity of lung and spleen immune responses in mice with slowly progressive primary tuberculosis. Scand J Immunol. 2000; 51: 147-154. doi: 10.1046/j.1365-3083.2000.00662.x. PMID: 10652161.
  46. Beltan E, Horgen L, Rastogi N. Secretion of cytokines by human macrophages upon infection by pathogenic and non-pathogenic mycobacteria. Microb Pathog. 2000; 28: 313-318. doi: 10.1006/ mpat.1999.0345. PMID: 10799281.
  47. Ragno S, Romano M, Howell S, Pappin DJ, Jenner PJ, Colston MJ. Changes in gene expression in macrophages infected with Mycobacterium tuberculosis: a combined transcriptomic and proteomic approach. Immunology. 2001; 104: 99-108. doi: 10.1046/ j.0019-2805.2001.01274.x. PMID: 11576227; PMCID: PMC1783284.
  48.   Kramer H, Bergmann KC. Zum Einfluss von Rifampicin auf die Lymphozytentransformation [The influence of rifampicin on the lymphocyte stimulation (author’s transl)]. Z Erkr Atmungsorgane. 1976; 144: 163-167. German. PMID: 969698.

Navolotskaya EV, Zinchenko DV, Kolobov AA, Murashev AN (2024) Action of Synthetic Peptide LKEKK in Experimental Tuberculosis. Clin Res Infect Dis 8(1): 1063.

Received : 06 Jun 2024
Accepted : 28 Jun 2024
Published : 01 Jul 2024
Journals
Annals of Otolaryngology and Rhinology
ISSN : 2379-948X
Launched : 2014
JSM Schizophrenia
Launched : 2016
Journal of Nausea
Launched : 2020
JSM Internal Medicine
Launched : 2016
JSM Hepatitis
Launched : 2016
JSM Oro Facial Surgeries
ISSN : 2578-3211
Launched : 2016
Journal of Human Nutrition and Food Science
ISSN : 2333-6706
Launched : 2013
JSM Regenerative Medicine and Bioengineering
ISSN : 2379-0490
Launched : 2013
JSM Spine
ISSN : 2578-3181
Launched : 2016
Archives of Palliative Care
ISSN : 2573-1165
Launched : 2016
JSM Nutritional Disorders
ISSN : 2578-3203
Launched : 2017
Annals of Neurodegenerative Disorders
ISSN : 2476-2032
Launched : 2016
Journal of Fever
ISSN : 2641-7782
Launched : 2017
JSM Bone Marrow Research
ISSN : 2578-3351
Launched : 2016
JSM Mathematics and Statistics
ISSN : 2578-3173
Launched : 2014
Journal of Autoimmunity and Research
ISSN : 2573-1173
Launched : 2014
JSM Arthritis
ISSN : 2475-9155
Launched : 2016
JSM Head and Neck Cancer-Cases and Reviews
ISSN : 2573-1610
Launched : 2016
JSM General Surgery Cases and Images
ISSN : 2573-1564
Launched : 2016
JSM Anatomy and Physiology
ISSN : 2573-1262
Launched : 2016
JSM Dental Surgery
ISSN : 2573-1548
Launched : 2016
Annals of Emergency Surgery
ISSN : 2573-1017
Launched : 2016
Annals of Mens Health and Wellness
ISSN : 2641-7707
Launched : 2017
Journal of Preventive Medicine and Health Care
ISSN : 2576-0084
Launched : 2018
Journal of Chronic Diseases and Management
ISSN : 2573-1300
Launched : 2016
Annals of Vaccines and Immunization
ISSN : 2378-9379
Launched : 2014
JSM Heart Surgery Cases and Images
ISSN : 2578-3157
Launched : 2016
Annals of Reproductive Medicine and Treatment
ISSN : 2573-1092
Launched : 2016
JSM Brain Science
ISSN : 2573-1289
Launched : 2016
JSM Biomarkers
ISSN : 2578-3815
Launched : 2014
JSM Biology
ISSN : 2475-9392
Launched : 2016
Archives of Stem Cell and Research
ISSN : 2578-3580
Launched : 2014
Annals of Clinical and Medical Microbiology
ISSN : 2578-3629
Launched : 2014
JSM Pediatric Surgery
ISSN : 2578-3149
Launched : 2017
Journal of Memory Disorder and Rehabilitation
ISSN : 2578-319X
Launched : 2016
JSM Tropical Medicine and Research
ISSN : 2578-3165
Launched : 2016
JSM Head and Face Medicine
ISSN : 2578-3793
Launched : 2016
JSM Cardiothoracic Surgery
ISSN : 2573-1297
Launched : 2016
JSM Bone and Joint Diseases
ISSN : 2578-3351
Launched : 2017
JSM Bioavailability and Bioequivalence
ISSN : 2641-7812
Launched : 2017
JSM Atherosclerosis
ISSN : 2573-1270
Launched : 2016
Journal of Genitourinary Disorders
ISSN : 2641-7790
Launched : 2017
Journal of Fractures and Sprains
ISSN : 2578-3831
Launched : 2016
Journal of Autism and Epilepsy
ISSN : 2641-7774
Launched : 2016
Annals of Marine Biology and Research
ISSN : 2573-105X
Launched : 2014
JSM Health Education & Primary Health Care
ISSN : 2578-3777
Launched : 2016
JSM Communication Disorders
ISSN : 2578-3807
Launched : 2016
Annals of Musculoskeletal Disorders
ISSN : 2578-3599
Launched : 2016
Annals of Virology and Research
ISSN : 2573-1122
Launched : 2014
JSM Renal Medicine
ISSN : 2573-1637
Launched : 2016
Journal of Muscle Health
ISSN : 2578-3823
Launched : 2016
JSM Genetics and Genomics
ISSN : 2334-1823
Launched : 2013
JSM Anxiety and Depression
ISSN : 2475-9139
Launched : 2016
Clinical Journal of Heart Diseases
ISSN : 2641-7766
Launched : 2016
Annals of Medicinal Chemistry and Research
ISSN : 2378-9336
Launched : 2014
JSM Pain and Management
ISSN : 2578-3378
Launched : 2016
JSM Women's Health
ISSN : 2578-3696
Launched : 2016
Clinical Research in HIV or AIDS
ISSN : 2374-0094
Launched : 2013
Journal of Endocrinology, Diabetes and Obesity
ISSN : 2333-6692
Launched : 2013
Journal of Substance Abuse and Alcoholism
ISSN : 2373-9363
Launched : 2013
JSM Neurosurgery and Spine
ISSN : 2373-9479
Launched : 2013
Journal of Liver and Clinical Research
ISSN : 2379-0830
Launched : 2014
Journal of Drug Design and Research
ISSN : 2379-089X
Launched : 2014
JSM Clinical Oncology and Research
ISSN : 2373-938X
Launched : 2013
JSM Bioinformatics, Genomics and Proteomics
ISSN : 2576-1102
Launched : 2014
JSM Chemistry
ISSN : 2334-1831
Launched : 2013
Journal of Trauma and Care
ISSN : 2573-1246
Launched : 2014
JSM Surgical Oncology and Research
ISSN : 2578-3688
Launched : 2016
Annals of Food Processing and Preservation
ISSN : 2573-1033
Launched : 2016
Journal of Radiology and Radiation Therapy
ISSN : 2333-7095
Launched : 2013
JSM Physical Medicine and Rehabilitation
ISSN : 2578-3572
Launched : 2016
Annals of Clinical Pathology
ISSN : 2373-9282
Launched : 2013
Annals of Cardiovascular Diseases
ISSN : 2641-7731
Launched : 2016
Journal of Behavior
ISSN : 2576-0076
Launched : 2016
Annals of Clinical and Experimental Metabolism
ISSN : 2572-2492
Launched : 2016
JSM Microbiology
ISSN : 2333-6455
Launched : 2013
Journal of Urology and Research
ISSN : 2379-951X
Launched : 2014
Journal of Family Medicine and Community Health
ISSN : 2379-0547
Launched : 2013
Annals of Pregnancy and Care
ISSN : 2578-336X
Launched : 2017
JSM Cell and Developmental Biology
ISSN : 2379-061X
Launched : 2013
Annals of Aquaculture and Research
ISSN : 2379-0881
Launched : 2014
Clinical Research in Pulmonology
ISSN : 2333-6625
Launched : 2013
Journal of Immunology and Clinical Research
ISSN : 2333-6714
Launched : 2013
Annals of Forensic Research and Analysis
ISSN : 2378-9476
Launched : 2014
JSM Biochemistry and Molecular Biology
ISSN : 2333-7109
Launched : 2013
Annals of Breast Cancer Research
ISSN : 2641-7685
Launched : 2016
Annals of Gerontology and Geriatric Research
ISSN : 2378-9409
Launched : 2014
Journal of Sleep Medicine and Disorders
ISSN : 2379-0822
Launched : 2014
JSM Burns and Trauma
ISSN : 2475-9406
Launched : 2016
Chemical Engineering and Process Techniques
ISSN : 2333-6633
Launched : 2013
Annals of Clinical Cytology and Pathology
ISSN : 2475-9430
Launched : 2014
JSM Allergy and Asthma
ISSN : 2573-1254
Launched : 2016
Journal of Neurological Disorders and Stroke
ISSN : 2334-2307
Launched : 2013
Annals of Sports Medicine and Research
ISSN : 2379-0571
Launched : 2014
JSM Sexual Medicine
ISSN : 2578-3718
Launched : 2016
Annals of Vascular Medicine and Research
ISSN : 2378-9344
Launched : 2014
JSM Biotechnology and Biomedical Engineering
ISSN : 2333-7117
Launched : 2013
Journal of Hematology and Transfusion
ISSN : 2333-6684
Launched : 2013
JSM Environmental Science and Ecology
ISSN : 2333-7141
Launched : 2013
Journal of Cardiology and Clinical Research
ISSN : 2333-6676
Launched : 2013
JSM Nanotechnology and Nanomedicine
ISSN : 2334-1815
Launched : 2013
Journal of Ear, Nose and Throat Disorders
ISSN : 2475-9473
Launched : 2016
JSM Ophthalmology
ISSN : 2333-6447
Launched : 2013
Journal of Pharmacology and Clinical Toxicology
ISSN : 2333-7079
Launched : 2013
Annals of Psychiatry and Mental Health
ISSN : 2374-0124
Launched : 2013
Medical Journal of Obstetrics and Gynecology
ISSN : 2333-6439
Launched : 2013
Annals of Pediatrics and Child Health
ISSN : 2373-9312
Launched : 2013
JSM Clinical Pharmaceutics
ISSN : 2379-9498
Launched : 2014
JSM Foot and Ankle
ISSN : 2475-9112
Launched : 2016
JSM Alzheimer's Disease and Related Dementia
ISSN : 2378-9565
Launched : 2014
Journal of Addiction Medicine and Therapy
ISSN : 2333-665X
Launched : 2013
Journal of Veterinary Medicine and Research
ISSN : 2378-931X
Launched : 2013
Annals of Public Health and Research
ISSN : 2378-9328
Launched : 2014
Annals of Orthopedics and Rheumatology
ISSN : 2373-9290
Launched : 2013
Journal of Clinical Nephrology and Research
ISSN : 2379-0652
Launched : 2014
Annals of Community Medicine and Practice
ISSN : 2475-9465
Launched : 2014
Annals of Biometrics and Biostatistics
ISSN : 2374-0116
Launched : 2013
JSM Clinical Case Reports
ISSN : 2373-9819
Launched : 2013
Journal of Cancer Biology and Research
ISSN : 2373-9436
Launched : 2013
Journal of Surgery and Transplantation Science
ISSN : 2379-0911
Launched : 2013
Journal of Dermatology and Clinical Research
ISSN : 2373-9371
Launched : 2013
JSM Gastroenterology and Hepatology
ISSN : 2373-9487
Launched : 2013
Annals of Nursing and Practice
ISSN : 2379-9501
Launched : 2014
JSM Dentistry
ISSN : 2333-7133
Launched : 2013
Author Information X